Neutralizing antibodies to influenza viruses

ABSTRACT

The present invention concerns methods and means for identifying, producing, and engineering neutralizing molecules against influenza A viruses, and to the neutralizing molecules produced. In particular, the invention concerns neutralizing molecules against various influenza A virus subtypes, including neutralizing antibodies against H5 and/or H3 and/or H1, such as, for example all of H1, H3, and H5 subtypes, and methods and means for making such molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under Section §119(e) and the benefitof U.S. Provisional Application Ser. No. 61/040,459 filed Mar. 28, 2008,the disclosure of which is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

The present invention concerns methods and means for identifying,producing, and engineering neutralizing molecules against viralantigens, including influenza A viruses, and to the neutralizingmolecules produced. The invention further concerns various uses of themolecules produced, including the design and production of vaccinesutilizing the binding sites of the neutralizing molecules of the presentinvention on the target viral antigen, such as influenza A virus.

Viruses are infectious pathogens that can cause serious diseasesincluding major threats for global public health, such as the influenza,AIDS, and hepatitis. A number of cancers have also been linked toviruses in conjunction with environmental factors. A typical virus is asub-micrometer particle that has DNA or RNA packaged in a shell known asthe capsid. Viral antigens protrude from the capsid and often fulfillimportant function in docking to the host cell, fusion, and injection ofviral DNA/RNA. Antibody-based immune responses form a first layer ofprotection of the host from viral infection, however, in many cases avigorous cellular immune response mediated by T-cells and NK-cells isrequired for effective viral clearance. When cellular immunity is unableto clear the virus, the infection can become chronic, and serumantibodies to the viral pathogen are used as first indicator for thediagnosis of the disease. Antibodies and antibody-like molecules wouldbe valuable tools for passive immunization against, or for the treatmentof such viral diseases.

One viral disease, the flu, is a contagious respiratory illness causedby influenza viruses. It causes mild to severe illness, and at times canlead to death. Annually, in the United States, influenza is contractedby 5-20% of the population, hospitalizing about 200,000, and causing thedeaths of about 36,000.

Influenza viruses spread in respiratory droplets caused by coughing andsneezing, which are usually transmitted from person to person. Immunityto influenza surface antigens, particularly hemagglutinin, reduces thelikelihood of infection and severity of disease if infection occurs.Although influenza vaccines are available, because a vaccine against oneinfluenza virus type or subtype confers limited or no protection againstanother type or subtype of influenza, it is necessary to incorporate oneor more new strains in each year's influenza vaccine.

Influenza viruses are segmented negative-strand RNA viruses and belongto the Orthomyxoviridae family. Influenza A virus consists of 9structural proteins and codes additionally for one nonstructural NS1protein with regulatory functions. The non-structural NS1 protein issynthesized in large quantities during the reproduction cycle and islocalized in the cytosol and nucleus of the infected cells. Thesegmented nature of the viral genome allows the mechanism of geneticreassortment (exchange of genome segments) to take place during mixedinfection of a cell with different viral strains. The influenza A virusmay be further classified into various subtypes depending on thedifferent hemagglutinin (HA) and neuraminidase (NA) viral proteinsdisplayed on their surface. Influenza A virus subtypes are identified bytwo viral surface glycoproteins, hemagglutinin (HA or H) andneuraminidase (NA or N). Each influenza virus subtype is identified byits combination of H and N proteins. There are 16 known HA subtypes and9 known NA subtypes. Influenza type A viruses can infect people, birds,pigs, horses, and other animals, but wild birds are the natural hostsfor these viruses. Only some influenza A subtypes (i.e., H1N1, H1N2, andH3N2) are currently in circulation among people, but all combinations ofthe 16H and 9 NA subtypes have been identified in avian species,especially in wild waterfowl and shorebirds. In addition, there isincreasing evidence that H5 and H7 influenza viruses can also causehuman illness.

The HA of influenza A virus comprises two structurally distinct regions,namely, a globular head region and a stem region. The globular headregion contains a receptor binding site which is responsible for virusattachment to a target cell and participates in the hemagglutinationactivity of HA. The stem region contains a fusion peptide which isnecessary for membrane fusion between the viral envelope and anendosomal membrane of the cell and thus relates to fusion activity(Wiley et al., Ann. Rev. Biochem., 56:365-394 (1987)).

A pandemic is a global disease outbreak. An influenza pandemic occurswhen a new influenza A virus: (1) emerges for which there is little orno immunity in the human population, (2) begins to cause seriousillness, and then (3) spreads easily person-to-person worldwide. Duringthe 20^(th) century there have been three such influenza pandemics.First, in 1918, the “Spanish Flu” influenza pandemic caused at least500,000 deaths in the United States and up to 40 million deathsworldwide. This pandemic was caused by influenza A H1N1 subtype. Second,in 1957, the “Asian Flu” influenza pandemic, caused by the influenza AH2N2 subtype, resulted in at least 70,000 deaths in the United Statesand 1-2 million deaths worldwide. Most recently in 1968 the “Hong KongFlu” influenza pandemic, caused by the influenza A H3N2 subtype,resulted in about 34,000 U.S. deaths and 700,000 deaths worldwide.

In 1997, the first influenza A H5N1 cases were reported in Hong Kong.This was the first time that this type of avian virus directly infectedhumans, but a pandemic did not result because human to humantransmission was not observed.

Lu et al., Resp. Res. 7:43 (2006) (doi: 10.1186/1465-992-7-43) reportthe preparation of anti-H5N1 IgGs from horses vaccinated withinactivated H5N1 virus, and of H5N1-specific F(ab′)₂ fragments, whichwere described to protect BALB/c mice infected with H5N1 virus.

Hanson et al., Resp. Res. 7:126 (doi: 10.1186/1465-9921-7-126) describethe use of a chimeric monoclonal antibody specific for influenza A H5virus hemagglutinin for passive immunization of mice.

Neutralizing antibodies to influenza viruses are disclosed in U.S.Application Publication No. 20080014205, published on Jan. 17, 2008.

In view of the severity of the respiratory illness caused by certaininfluenza A viruses, and the threat of a potential pandemic, there is agreat need for effective preventative and treatment methods. The presentinvention addresses this need by providing influenza A neutralizingmolecules against various H subtypes of the virus, including, withoutlimitation, the H1, and H3 subtypes, and the H5 subtype of the influenzaA virus. The invention further provides molecules capable ofneutralizing more than one, and preferably all, isolates (strains) of agiven subtype of the influenza A virus, including, without limitation,isolates obtained from various human and non-human species and isolatesfrom victims and/or survivors of various influenza epidemics and/orpandemics.

Such neutralizing molecules can be used for the prevention and/ortreatment influenza virus infection, including passive immunization ofinfected or at risk populations in cases of epidemics or pandemics.

SUMMARY OF THE INVENTION

In one aspect, the present invention concerns a molecule that canneutralize at least one subtype of an influenza virus and/or at leastone isolate of an influenza virus. In one embodiment, the molecule is anantibody or an antibody-like molecule, wherein the molecule (i)neutralizes more than one subtype and/or more than one isolate of aninfluenza A virus, (ii) binds to a hemagglutinin (HA) antigen of thevirus, and (iii) does not inhibit hemagglutination. In anotherembodiment, the molecule is a polypeptide comprising a VpreB sequenceand/or a λ5 sequence. In one other embodiment, the molecule is apolypeptide comprising a VpreB sequence fused to a λ5 sequence. Inanother embodiment, the molecule is a κ-like surrogate light chain (SLC)construct comprising a Vκ-like and/or a JCκ sequence. In one embodiment,the molecule is an antibody. In another embodiment, the molecule iscross-reactive with at least two HA antigens selected from the groupconsisting of H1, H2, H3, H5, H6, H7, H8 and H9. In yet anotherembodiment, the molecule is cross-reactive with at least two HA antigensselected from the group consisting of H1, H2, H3, H5, and H9.

In all embodiments, the molecule is an antibody or an antibody-likemolecule.

In all embodiments, the molecule is an antibody fragment.

In one aspect, the present invention concerns a neutralizing moleculeneutralizing an influenza A virus subtype. In one embodiment, themolecule is an antibody or an antibody-like molecule, wherein themolecule (i) neutralizes more than one subtype and/or more than oneisolate of an influenza A virus, (ii) binds to a hemagglutinin (HA)antigen of the virus, and (iii) does not inhibit hemagglutination. Inother embodiments, the molecule does not prevent the influenza A virus'globular head region from binding the surface of a cell. In anotherembodiment, the cell is a cell to be infected. In another embodiment,the molecule does not prevent the influenza A virus from attaching to acell

In one embodiment, the molecule binds to an epitope of an H5 subtype ofthe HA antigen; an H1 subtype of the HA antigen; or an H3 subtype of theHA antigen. In one other embodiment, the H5, H3, or H1 epitope isdisplayed on the surface of an influenza A virus. In another embodiment,the H5 subtype is an H5N1 subtype. In one other embodiment, the H1subtype is an H1N1 subtype. In another embodiment, the moleculeneutralizes more than one isolate of the H5 and/or H3 and/or H1influenza A virus subtypes. In another embodiment, the moleculeneutralizes more than one isolate of the H5N1 and/or H3N1 and/or H1N1influenza A virus subtypes. In one embodiment, the molecule neutralizesall isolates of the H5 and/or H3 and/or H1 influenza A virus subtypes.

In all embodiments, the influenza A virus subtypes that are neutralizedmay be further characterized by a neuraminidase (N) glycoprotein,including without limitation N1 and N2.

In one other embodiment, the molecule neutralizes at least an H5 and/orH3 and/or an H1 influenza A virus subtypes.

In another embodiment, the molecule neutralizes all H5 and/or H3 and/orH1 influenza A virus subtypes.

In another embodiment, the molecule neutralizes more than one H5 and/orH3 and/or H1 isolate of an influenza A virus subtype.

In yet another embodiment, the molecule neutralizes all H5 and/or H3and/or H1 isolates of an influenza A virus subtype.

In another embodiment, the molecule neutralizes all H5 and/or H3 and/orH1 isolates of an influenza A virus subtype where the isolates arecapable of infecting humans.

In all embodiments, the H5 subtype may comprise an H5 antigen and/or theH1 subtype may comprise an H1 antigen.

In one other embodiment, the present invention provides a molecule whichbinds essentially the same epitope as the epitope for a molecule havinga heavy chain polypeptide containing an amino acid sequence shown as SEQID NO:4, SEQ ID NO:45, SEQ ID NO:9, or SEQ ID NO:61; or a consensus orvariant sequence based upon said amino acid sequences, or a fragmentthereof. In another embodiment, the molecule binds essentially the sameepitope as the epitope for a molecule comprising a light chainpolypeptide containing an amino acid sequence shown as SEQ ID NO:71, SEQID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:160;or a consensus or variant sequence based upon said amino acid sequences.In some embodiments, the present invention provides a moleculecomprising a heavy chain polypeptide containing SEQ ID NO:4, SEQ IDNO:45, SEQ ID NO:9, or SEQ ID NO:61, or a consensus or variant sequencebased upon said amino acid sequences, or a fragment thereof. In otherembodiments, the molecule further contains a light chain polypeptidecontaining SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQID NO:159, or SEQ ID NO:160, or a consensus or variant sequence basedupon said amino acid sequences, or a fragment thereof.

In one embodiment, the molecule binds essentially the same epitope as amolecule that includes a heavy chain polypeptide containing an aminoacid sequence having the formula:X₁-X₂-Q-L-V-Q-S-G-X₃-E-V-X₄-K-P-G-X₅-S-V-X₆-X₇-S-C-K-X₈-S-G-G-X₉-F-S-S-Y-A-X₁₀-X₁₁-W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X₁₂-G-I-I-X₁₃-X₁₄-F-G-T-T-X₁₅-N-Y-A-Q-K-F-Q-G-R-X₁₆-T-X₁₇-T-A-D-X₁₉-X₁₉-T-S-T-A-Y-M-E-L-S-S-L-R-S-X₂₀-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X₂₁-X₂₂-L-D-Y-W-G-X₂₃-G-T-X₂₄(SEQ ID NO:161), or a consensus or variant sequence based upon saidamino acid sequences; wherein X₁ is Q or E; X₂ is V or M; X₃ is A or T;X₄ is K or Q; X₅ is S or A; X₆ is K or R; X₇ is V or L; X₈ is A, T or V;X₉ is T, S or A; X₁₀ is I or V; X₁₁ is S or T; X₁₂ is G or A; X₁₃ is Por G; X₁₄ is I or M; X₁₅ is A or T; X₁₆ is V or L; X₁₇ is I, L, or M;X₁₈ is K or E; X₁₉ is S, L or M; X₂₀ is E or D; X₂₁ is S, T or N; X₂₂ isS or T; X₂₃ is Q, K, G or R; and X₂₄ is L, T or M. In one embodiment,the amino acid sequence shown as SEQ ID NO:161 further comprises-V-T-V-S-S or -V-R-V-S-S at the C-terminal end following X₂₄.

In yet another embodiment, the molecule binds essentially the sameepitope as the epitope for a molecule containing a light chainpolypeptide containing an amino acid sequence shown as SEQ ID NO:71, SEQID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:160,or a consensus or variant sequence based upon said amino acid sequences.

In some embodiments, the present invention provides a moleculecomprising a heavy chain polypeptide containing a heavy chainpolypeptide containing an amino acid sequence having the formula:X₁-X₂-Q-L-V-Q-S-G-X₃-E-V-X₄-K-P-G-X₅-S-V-X₆-X₇-S-C-K-X₈-S-G-G-X₉-F-S-S-Y-A-X₁₀-X₁₁-W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X₁₂-G-I-I-X₁₃-X₁₄-F-G-T-T-X₁₅-N-Y-A-Q-K-F-Q-G-R-X₁₆-T-X₁₇-T-A-D-X₁₈-X₁₉-T-S-T-A-Y-M-E-L-S-S-L-R-S-X₂₀-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X₂₁-X₂₂-L-D-Y-W-G-X₂₃-G-T-X₂₄(SEQ ID NO:161), or a consensus or variant sequence based upon saidamino acid sequences; wherein X₁ is Q or E; X₂ is V or M; X₃ is A or T;X₄ is K or Q; X₅ is S or A; X₆ is K or R; X₇ is V or L; X₈ is A, T or V;X₉ is T, S or A; X₁₀ is I or V; X₁₁ is S or T; X₁₂ is G or A; X₁₃ is Por G; X₁₄ is I or M; X₁₅ is A or T; X₁₆ is V or L; X₁₇ is I, L, or M;X₁₈ is K or E; X₁₉ is S, L or M; X₂₀ is E or D; X₂₁ is S, T or N; X₂₂ isS or T; X₂₃ is Q, K, G or R; and X₂₄ is L, T or M. In one embodiment,the amino acid sequence shown as SEQ ID NO:161 further comprises-V-T-V-S-S or -V-R-V-S-S at the C-terminal end following X₂₄.

In other embodiments, the molecule further contains a light chainpolypeptide containing an amino acid sequence shown as SEQ ID NO:71, SEQID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159, or SEQ ID NO:160.

In all embodiments, the molecule is an antibody or an antibody-likemolecule.

In one embodiment, the antibody or an antibody-like molecule (i)neutralizes more than one subtype and/or more than one isolate of aninfluenza A virus, (ii) binds to a hemagglutinin (HA) antigen of thevirus, and (iii) does not inhibit hemagglutination.

In another embodiment, at least one of the virus subtypes and/orisolates neutralized by the molecules herein has the ability to infecthumans.

In other embodiments, at least one of said isolates has been obtainedfrom a human subject. In another embodiment, the human subject is or wasdiseased with an influenza virus at the time of obtaining the isolate.In other embodiments, the human subject recovered from infection withthe influenza virus A. In another embodiment, the influenza virus A isan H5 subtype and/or an H1 subtype of influenza virus A.

In one embodiment, at least one of said isolates has been obtained froma non-human animal. In another embodiment, at least one of the isolatesis from a bird, including, without limitation, wild-fowls and chicken.

In one embodiment, the molecules neutralize an H5 subtype and an H1subtype.

In another embodiment, the neutralizing molecules of the presentinvention bind the H5 and/or H1 protein. In one embodiment, the H5protein is an H5 HA protein. Preferably, the molecules bind more thanone variant of the H5 protein, or, even more preferably, substantiallyall variants of the H5 protein.

In another embodiment, the molecule also binds to at least oneadditional HA antigen. In one other embodiment, the additional HAantigen is an H1 HA antigen. In one other embodiment, the molecule bindsto substantially all variants of the H1 HA protein. In one embodiment,the at least one additional HA antigen is selected from the groupconsisting of H2, H3, H5, H6, H7, H8 and H9. In another embodiment, theat least one additional HA antigen also binds to HA antigen H5; HAantigens H3 and H9; or HA antigens H3, H5, and H9.

In other embodiments, the molecules described herein bind to the H5protein and to at least one additional H protein, such as an H1 protein.

In a different aspect, the invention concerns compositions comprisingthe neutralizing molecules described herein. In one embodiment, thecompositions comprise an antibody or antibody-like molecule describedherein.

In a further aspect, the invention concerns a method for identifying amolecule capable of neutralizing more than one isolate of a singleinfluenza A virus subtype or multiple influenza A virus subtypes. Thismethod comprises identifying molecules, e.g., antibodies, antibodyfragments, or antibody-like molecules in an antibody library, that reactwith both a first and a second isolate of the influenza A virus subtypeor with a first and a second subtype of the influenza A virus, andsubjecting the molecules identified to successive, alternating rounds ofselection, based on their ability to bind the first and second isolates,or the first and second subtypes, respectively. In one embodiment, themethod further comprises isolating the identified antibody.

In another embodiment, molecules that react with both a first and asecond influenza A virus subtype isolate have been identified by atleast two rounds of separate enrichment of molecules reacting with thefirst isolate and the second isolate, respectively, and recombining themolecules identified.

In another aspect, the present invention provides a method ofidentifying an antibody capable of neutralizing an isolate of an H5influenza A virus and/or an isolate of an H1 influenza A virus; or asubtype of an H5 influenza A virus and/or a subtype of an H1 influenza Avirus. In one embodiment, the method comprises identifying, in anantibody library, antibodies that react with both an H5 isolate and/oran H1 isolate; or an H5 subtype and/or an H1 subtype, and subjecting theantibodies identified to successive alternating rounds of selection,based on their ability to bind said H5 and/or H1 isolates or HAproteins; or said H5 and/or H1 subtypes or HA proteins, respectively. Inanother embodiment, the method comprises at least two rounds ofselection. In one embodiment, the method further comprises isolating theidentified antibody. In another embodiment, the H5 isolate is an H5subtype of said influenza A virus or HA and/or said H1 isolate is an H1subtype of said influenza A virus or HA. In yet another embodiment, theantibodies that react with both a first and a second influenza A virussubtype isolate or HA have been identified by at least two rounds ofseparate enrichment of antibodies reacting with the first isolate or HAand the second isolate or HA, respectively, and recombining theantibodies identified. In one other embodiment, the antibody that canreact with both said H5 and said H1 influenza A subtype isolates or HAsis subjected to mutagenesis prior to being subjected to said successivealternating rounds of selection, based on their ability to bind said H5and second H1 subtype isolates or HAs, respectively. In one otherembodiment, the influenza A virus subtype is an H5 subtype or HA andsaid influenza A virus subtype is an H1 subtype or HA. In anotherembodiment, the H5 subtype is, or the HA is from, a 2006 Turkish isolateof the H5 virus; the H5 subtype is, or the HA is from, a 2003/2004Vietnam isolate of the H5 virus; the H5 subtype is, or the HA is from, a1997 Hong Kong isolate of the H5 virus; the H1 subtype is, or the HA isfrom, a New Calcdonia/20/99 isolate of the H1 virus; the H5 and/or saidH1 subtypes or HAs originate from different species; or any combinationthereof. In one other embodiment, at least one of said species is human;or at least one of said species is a bird. In another embodiment, theantibodies capable of binding said H5 and/or said H1 isolates areadditionally selected based on their ability to bind more than oneinfluenza A subtype.

In another embodiment, the molecule library is a phage display library.In one embodiment, the selection is performed by biopanning.

In another embodiment, the molecule that can react with both the firstand the second influenza A subtype isolate is subjected to mutagenesisprior to being subjected to successive alternating rounds of selection,based on its ability to bind the first and second isolate, respectively.If desired, the molecules capable of binding the first and the secondisolate are additionally selected based on their ability to bind morethan one influenza A subtype.

The application of such enrichment techniques can be similarly appliedto molecules in general, regardless of the target to which they bind.Such general enrichment/selection methods are specifically included aspart of the invention.

In another embodiment, the invention concerns a collection of sequencesshared by the neutralizing molecules of the present invention andidentified by the methods described herein. In one other embodiment, thecollection of sequences comprises one or more of the unique heavy and/orlight chain sequences shown in Table 2 or a consensus or variantsequence based on said sequences. In another embodiment, the presentinvention provides a neutralizing antibody or a fragment thereof,identified by the methods described herein.

In a still further aspect, the invention concerns a method for treatingan influenza A infection in a subject comprising of administering to thesubject an effective amount of a neutralizing molecule or moleculecomposition herein.

In another aspect, the invention concerns a method for preventinginfluenza A infection comprising of administering to a subject at riskof developing influenza A infection an effective amount of aneutralizing molecule or molecule composition described herein. In oneembodiment, the neutralizing molecule is a neutralizing antibody,antibody fragment, or antibody-like molecule.

In all embodiments, the subject is a human patient. In all embodiments,the subject is a subject at risk of developing an influenza A infection.

In a different aspect, the invention concerns a method for producing adiverse multifunctional molecule collection, comprising: (a) aligningCDR sequences of at least two functionally different molecules, e.g.,antibodies, antibody fragments, or antibody-like molecules, (b)identifying amino acid residues conserved between the CDR sequencesaligned, and (c) performing mutagenesis of multiple non-conserved aminoacid residues in at least one of the CDR sequences aligned, usingdegenerate oligonucleotide probes encoding at least the amino acidresidues present in the functionally different molecules at thenon-conserved positions mutagenized to produce multiple variants of thealigned CDR sequences, and, if desired, repeating steps (b) and (c) withone or more of the variants until the molecule collection reaches adesired degree of diversity and/or size.

In a particular embodiment, the CDR sequences aligned have the samelengths.

In another embodiment, the conserved amino acid residues are retained inat least two of the CDR sequences aligned.

In a further aspect, the invention concerns a molecule collectioncomprising a plurality of neutralizing molecules, e.g., antibodies,antibody fragments, or antibody-like molecules, which differ from eachother in at least one property.

The invention further concerns a method for uniquely identifying nucleicacids in a collection comprising labeling the nucleic acids with aunique barcode linked to or incorporated in the sequences of the nucleicacid present in such collection.

The invention further concerns a vaccine effective against influenza Avirus containing a peptide or polypeptide that functionally mimics aneutralization epitope bound by a molecule of the present invention. Inone embodiment, the vaccine is a synthetic vaccine. In anotherembodiment, the vaccine contains an attenuated influenza A virus, or apart thereof. In one other embodiment, the vaccine contains a killedinfluenza A virus, or part thereof. In another embodiment, the moleculethat binds a neutralization epitope is one of the following:

(a) a molecule which (i) neutralizes more than one subtype and/or morethan one isolate of an influenza A virus, (ii) binds to a hemagglutinin(HA) antigen of the virus, and (iii) does not prevent hemagglutination;

(b) a molecule which binds essentially the same epitope as the epitopefor a molecule comprising a heavy chain polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:4, SEQ IDNO:45, SEQ ID NO:9, and SEQ ID NO:61; or a consensus or variant sequencebased upon said amino acid sequences, or a fragment thereof;

(c) a molecule which binds essentially the same epitope as the epitopefor a molecule comprising a heavy chain polypeptide comprising an aminoacid sequence having the formula:X₁-X₂-Q-L-V-Q-S-G-X₃-E-V-X₄-K-P-G-X₅-S-V-X₆-X₇-S-C-K-X₈-S-G-G-X₉-F-S-S-Y-A-X₁₀-X₁₁-W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X₁₂-G-I-I-X₁₃-X₁₄-F-G-T-T-X₁₅-N-Y-A-Q-K-F-Q-G-R-X₁₆-T-X₁₇-T-A-D-X₁₈-X₁₉-T-S-T-A-Y-M-E-L-S-S-L-R-S-X₂₀-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X₂₁-X₂₂-L-D-Y-W-G-X₂₃-G-T-X₂₄(SEQ ID NO:161); or a consensus or variant sequence based upon saidamino acid sequences, or a fragment thereof; wherein X₁ is Q or E; X₂ isV or M; X₃ is A or T; X₄ is K or Q; X₅ is S or A; X₆ is K or R; X₇ is Vor L; X₈ is A, T or V; X₉ is T, S or A; X₁₀ is I or V; X₁₁ is S or T;X₁₂ is G or A; X₁₃ is P or G; X₁₄ is I or M; X₁₅ is A or T; X₁₆ is V orL; X₁₇ is I, L, or M; X₁₈ is K or E; X₁₉ is S, L or M; X₂₀ is E or D;X₂₁ is S, T or N; X₂₂ is S or T; X₂₃ is Q, K, G or R; and X₂₄ is L, T orM;

(d) a molecule comprising a heavy chain polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:4, SEQ IDNO:45, SEQ ID NO:9, and SEQ ID NO:61; or a consensus or variant sequencebased upon said amino acid sequences, or a fragment thereof; or

(e) a molecule comprising a heavy chain polypeptide comprising an aminoacid sequence having the formula:X₁-X₂-Q-L-V-Q-S-G-X₃-E-V-X₄-K-P-G-X₅-S-V-X₆-X₇-S-C-K-X₈-S-G-G-X₉-F-S-S-Y-A-X₁₀-X₁₁-W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X₁₂-G-I-I-X₁₃-X₁₄-F-G-T-T-X₁₅-N-Y-A-Q-K-F-Q-G-R-X₁₆-T-X₁₇-T-A-D-X₁₈-X₁₉-T-S-T-A-Y-M-E-L-S-S-L-R-S-X₂₀-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X₂₁-X₂₂-L-D-Y-W-G-X₂₃-G-T-X₂₄(SEQ ID NO:161); or a consensus or variant sequence based upon saidamino acid sequences, or a fragment thereof; wherein X₁ is Q or E; X₂ isV or M; X₃ is A or T; X₄ is K or Q; X₅ is S or A; X₆ is K or R; X₇ is Vor L; X₈ is A, T or V; X₉ is T, S or A; X₁₀ is I or V; X₁₁ is S or T;X₁₂ is G or A; X₁₃ is P or G; X₁₄ is I or M; X₁₅ is A or T; X₁₆ is V orL; X₁₇ is I, L, or M; X₁₈ is K or E; X₁₉ is S, L or M; X₂₀ is E or D;X₂₁ is S, T or N; X₂₂ is S or T; X₂₃ is Q, K, G or R; and X₂₄ is L, T orM.

In one embodiment, the amino acid sequence shown as SEQ ID NO:161further comprises -V-T-V-S-S or -V-R-V-S-S at the C-terminal endfollowing X₂₄.

In another embodiment, the vaccine is based on a molecule that binds anHA antigen. In some other embodiments, the HA antigen is an H5 subtypeor an H1 subtype. In one other embodiment, the antigen is displayed onthe surface of an influenza A virus. In yet another embodiment, thepeptide or polypeptide contains antigenic determinants that raiseneutralizing molecules, e.g., antibodies.

In all embodiments, the present invention provides compositions thatcomprise a molecule described herein. In all embodiments, the moleculeis an antibody, antibody fragment, or antibody-like molecule.

In one aspect, the present invention provides neutralizing antibodiesidentified by the methods described herein. In one embodiment, theneutralizing antibody is an antibody or an antibody fragment. In anotherembodiment, the neutralizing antibody or antibody fragment is capable ofconferring passive immunity to an avian or mammalian subject against aninfluenza A virus infection. In another embodiment, the mammaliansubject is a human. In one other embodiment, the influenza A virusinfection is caused by an H5 subtype and/or an H1 subtype.

In another aspect, the present invention provides molecules capable ofbinding to and neutralizing a viral antigen. In one embodiment, themolecule comprises an antibody heavy chain variable domain comprising atleast one substitution in the surface exposed cluster determined byamino acid positions 52A, 53, 73, and 74, following Kabat amino acidnumbering, wherein said molecule is capable of binding to andneutralizing a viral antigen. In another embodiment, the moleculecomprises a substitution at least one of amino acid positions 52A, 53,73, and 74. In another embodiment, the molecule comprises a substitutionat all of amino acid positions 52A, 53, 73, and 74. In anotherembodiment, the molecule further comprises a substitution at amino acidposition 57. In another embodiment, the molecule further comprises P52G,I53M, L73E, and S74L/M substitutions. In another embodiment, themolecule additionally comprises an A57T substitution. In anotherembodiment, the molecule also comprises a substitution at least one ofamino acid positions 24, 34, 35 and 50. In another embodiment, themolecule comprises substitutions at all of amino acid positions 24, 34,35 and 50. In another embodiment, the molecule comprises V24T, W34V,G35T and S50A substitutions.

In one aspect, the molecules of the present invention comprise a heavychain variable domain sequence from a germ-line heavy chain. In oneembodiment, the germ-line heavy chain is a V_(H)1e or a V_(H)1-69germ-line heavy chain. In another embodiment, the rest of the heavychain variable domain sequence retains the sequence of the germ-lineheavy chain. In another embodiment, the germ-line heavy chain variabledomain comprises at least one additional conservative substitution.

In one embodiment, the molecules further comprise a light chainsequence. In another embodiment, the light chain sequence is an antibodyλ or κ light chain sequence. In one embodiment, the light chain sequenceis a surrogate light chain sequence. In one other embodiment, thesurrogate light chain sequence comprises a VpreB sequence and/or a λ5sequence. In yet another embodiment, the surrogate light chain sequencecomprises a VpreB sequence fused to a λ5 sequence. In anotherembodiment, the surrogate light chain sequence is a κ-like surrogatelight chain (SLC) construct comprising a Vκ-like and/or a JCκ sequence.

In one embodiment, the viral antigen neutralized by the molecule isselected from the group consisting of viral antigens from influenzaviruses, HIV-1, HIV-2, HTLV-I and -II viruses, SARS coronavirus, herpessimplex virus, Epstein Barr virus, cytomegalovirus, hepatitis virus(HCV, HAV, HBV, HDV, HEV), toxoplasma gondii virus, treponema pallidiumvirus, human T-lymphotrophic virus, encephalitis virus, West Nile virus,Dengue virus, Varicella Zoster Virus, rubeola, mumps, and rubella.

In another embodiment, the viral antigen is from an influenza virus oran HIV-1 or HIV-2 virus.

In one other aspect, the present invention provides vaccines effectiveagainst influenza A virus. In one embodiment, the vaccine comprises apeptide or polypeptide functionally mimicking a neutralization epitopeof a molecule described herein. In another embodiment, the vaccineeffective against a viral antigen comprises a peptide or polypeptidefunctionally mimicking a neutralization epitope of a molecule describedherein. In one embodiment, the viral antigen is from an influenza virusor an HIV-1 or HIV-2 virus.

In another embodiment, the vaccine is a vaccine effective against aninfluenza A virus, comprising a peptide or polypeptide functionallymimicking a neutralization epitope of a molecule described herein. Inone embodiment, the molecule is an antibody. In another embodiment, theantibody binds an HA antigen. In one other embodiment, the HA antigen isan H5 subtype. In one other embodiment, the HA antigen is an H1 subtype.In one other embodiment, the antigen is displayed on the surface of aninfluenza A virus. In one other embodiment, the peptide or polypeptidecomprises antigenic determinants that raise neutralizing antibodies.

In one embodiment, the vaccine is a synthetic vaccine. In anotherembodiment, the vaccine comprises an attenuated influenza A virus, or apart thereof. In one other embodiment, the vaccine comprises a killedinfluenza A virus, or part thereof.

In other embodiments, the vaccine is suitable for oral administration,parenteral administration, transdermal delivery, or transmucosaldelivery. In one embodiment, the transmucosal delivery is intra-nasaladministration. In one other embodiment, the vaccine is for childhoodimmunization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a typical panning enrichment scheme for increasingthe reactive strength towards two different targets, A and B. Each roundof enrichment increases the reactive strength of the pool towards theindividual target(s).

FIG. 2 illustrates a strategy for the selection of clones cross-reactivewith targets A and B, in which each successive round reinforces thereactive strength of the resulting pool towards both targets.

FIG. 3 illustrates a strategy for increasing the reactive strengthstowards two different targets (targets A and B), by recombining paralleldiscovery pools to generate/increase cross-reactivity. Each round ofselection of the recombined antibody library increases the reactivestrength of the resulting pool towards both targets.

FIG. 4 illustrates a strategy for increasing cross-reactivity to atarget B while maintaining reactivity to a target A. First, a clonereactive with target A is selected, then a mutagenic library of theclones reactive with target A is prepared, and selection is performed asshown, yielding one or more antibody clones that show strong reactivitywith both target A and target B.

FIG. 5 illustrates a representative mutagenesis method for generating adiverse multifunctional antibody collection by the “destinationalmutagenesis” method.

FIG. 6 shows the analysis of antibody binding to hemagglutinins fromdifferent influenza A subtypes.

FIG. 7 shows the positions of H5 hemagglutinin binding Group 1 requiredand dominant mutations on the crystal structure of Fab 47e.

FIG. 8 shows the cross-reactive titers of Turkish avian influenzasurvivors to the H5N1 Vietnam 1203/04 hemagglutinin protein.

FIG. 9 illustrates the cloning and barcoding of annotated repertoires.

DETAILED DESCRIPTION A. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al., Dictionary ofMicrobiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York,N.Y. 1994), provides one skilled in the art with a general guide to manyof the terms used in the present application.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described. For purposes ofthe present invention, the following terms are defined below.

The terms “influenza A subtype” or “influenza A virus subtype” are usedinterchangeably, and refer to influenza A virus variants that arecharacterized by a hemagglutinin (H) viral surface protein, and thus arelabeled by an H number, such as, for example, H1, H3, and H5. Inaddition, the subtypes may be further characterized by a neuraminidase(N) viral surface protein, indicated by an N number, such as, forexample, N1 and N2. As such, a subtype may be referred to by both H andN numbers, such as, for example, H1N1, H5N1, and H5N2. The termsspecifically include all strains (including extinct strains) within eachsubtype, which usually result from mutations and show differentpathogenic profiles. Such strains will also be referred to as various“isolates” of a viral subtype, including all past, present and futureisolates. Accordingly, in this context, the terms “strain” and “isolate”are used interchangeably. Subtypes contain antigens based upon aninfluenza A virus. The antigens may be based upon a hemagglutinin viralsurface protein and can be designated as “HA antigen”. In someinstances, such antigens are based on the protein of a particularsubtype, such as, for example, an H1 subtype and an H5 subtype, whichmay be designated an H1 antigen and an H5 antigen, respectively.

The term “influenza” is used to refer to a contagious disease caused byan influenza virus.

In the context of the present invention, the term “antibody” (Ab) isused in the broadest sense and includes polypeptides which exhibitbinding specificity to a specific antigen.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby covalent disulfide bond(s), while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy and light chain also has regularly spaced intrachaindisulfide bridges. Each heavy chain has, at one end, a variable domain(V_(H)) followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains, Chothia et al., J. Mol. Biol. 186:651(1985); Novotny and Haber, Proc. Natl. Acad. Sci. U.S.A. 82:4592 (1985).

The term “variable” with reference to antibody chains is used to referto portions of the antibody chains which differ extensively in sequenceamong antibodies and participate in the binding and specificity of eachparticular antibody for its particular antigen. Such variability isconcentrated in three segments called hypervariable regions both in thelight chain and the heavy chain variable domains. The more highlyconserved portions of variable domains are called the framework region(FR). The variable domains of native heavy and light chains eachcomprise four FRs (FR1, FR2, FR3 and FR4, respectively), largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), pages 647-669). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e., residues 30-36(L1), 46-55 (L2) and 86-96 (L3) in the light chain variable domain and30-35 (H1), 47-58 (H2) and 93-101 (H3) in the heavy chain variabledomain; MacCallum et al., J Mol. Biol. 1996. “Framework” or “FR”residues are those variable domain residues other than the hypervariableregion residues as herein defined.

Depending on the amino acid sequence of the constant domain of theirheavy chains, antibodies can be assigned to different classes. There arefive major classes of antibodies IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

The heavy-chain constant domains that correspond to the differentclasses of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (κ) andlambda (λ), based on the amino acid sequences of their constant domains.

The term “antibody fragment” is a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,and Fv fragments, linear antibodies, single-chain antibody molecules,diabodies, and multispecific antibodies formed from antibody fragments.Further examples of antibody fragments include, but are not limited to,scFv, (scFv)₂, dAbs (single-domain antibodies), and complementaritydetermining region (CDR) fragments, and minibodies, which are minimizedvariable domains whose two loops are amenable to combinatorialmutagenesis.

The term “monoclonal antibody” is used to refer to an antibody moleculesynthesized by a single clone of B cells. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. Thus, monoclonal antibodies may be made by the hybridoma methodfirst described by Kohler and Milstein, Nature 256:495 (1975); Eur. J.Immunol. 6:511 (1976), by recombinant DNA techniques, or may also beisolated from phage antibody libraries.

The term “polyclonal antibody” is used to refer to a population ofantibody molecules synthesized by a population of B cells.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains which enables thesFv to form the desired structure for antigen binding. For a review ofsFv see Plückthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994). Single-chain antibodies are disclosed, for example in WO88/06630 and WO 92/01047.

The term “diabody” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

The term “minibody” is used to refer to an scFv-CH3 fusion protein thatself-assembles into a bivalent dimer of 80 kDa (scFv-CH3)₂.

The term “aptamer” is used herein to refer to synthetic nucleic acidligands that bind to protein targets with high specificity and affinity.Aptamers are known as potent inhibitors of protein function.

A dAb fragment (Ward et al., Nature 341:544 546 (1989)) consists of aV_(H) domain or a V_(L) domain.

As used herein the term “antibody binding regions” refers to one or moreportions of an immunoglobulin or antibody variable region capable ofbinding an antigen(s). Typically, the antibody binding region is, forexample, an antibody light chain (V_(L)) (or variable region thereof),an antibody heavy chain (V_(H)) (or variable region thereof), a heavychain Fd region, a combined antibody light and heavy chain (or variableregion thereof) such as a Fab, F(ab′)₂, single domain, or single chainantibody (scFv), or a full length antibody, for example, an IgG (e.g.,an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgMantibody.

The term “bispecific antibody” refers to an antibody that showsspecificities to two different types of antigens. The term as usedherein specifically includes, without limitation, antibodies which showbinding specificity for a target antigen and to another target thatfacilitates delivery to a particular tissue. Similarly, multi-specificantibodies have two or more binding specificities.

The expression “linear antibody” is used to refer to comprising a pairof tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific and are described, for example, by Zapata et al., ProteinEng 8(10):1057-1062 (1995).

For the purposes of the present invention, the term “antibody-likemolecule” includes any molecule, other than an antibody fragment ashereinabove defined, that is capable of binding to and neutralizing aviral antigen. The term specifically includes, without limitation, pre-Bcell receptor (pre-BCR) like structures, referred to as “surrobodies,”including surrogate light chain (SLC) elements, as described, forexample, in PCT Publication No. WO 2008/118970, published Oct. 2, 2008,and in Xu et al. Proc. Natl. Acad. Sci. USA, 105(31): 10756-61 (2008).The SLC is a nondiversified heterodimer composed of the noncovalentlyassociated Vpre-B and λ5 proteins. The VpreB chain is homologous to a VλIg domain, and the λ5 chain is homologous to the Cλ domain of canonicalantibodies, respectively. The heterodimeric SLC is covalently associatedwith the heavy chain in the pre-BCR complex by disulfide bonds betweenthe Cλ domain and the first constant domain of the pre-BCR HC. A uniquefeature of the SLC is that the VpreB1 and the λ5 domains each havenoncanonical peptide extensions. VpreB1 has an additional 21 residues onits C terminus, and λ5 has a 50-aa-long tail on its N terminus (see,e.g. Vettermann et al., Semin. Immunol. 18:44-55 (2006)). The surrobodystructures specifically include, without limitation, the native trimericpre-BCR-like functional unit of the pre-BCR, fusion of VpreB1 to λ5, andtrimers that eliminated either the λ5 N-terminal 50 aa or the VpreB1C-terminal 21 aa or both peptide extensions. In addition, chimericconstructs using the constant components of classical antibody lightchains are specifically included within the definition of surrobodies.

Other representatives of “antibody-like molecules,” as defined herein,are similar structures comprising antibody surrogate κ light chainsequences, where κ light chain sequences are optionally partnered withanother polypeptide, such as, for example, antibody heavy and/or lightchain domain sequences. A κ-like B cell receptor (κ-like BCR) has beenidentified, utilizing a κ-like surrogate light chain (κ-like SLC)(Frances et al., EMBO J 13:5937-43 (1994); Thompson et al.,Immunogenetics 48:305-11 (1998); Rangel et al., J Biol Chem 280:17807-14(2005)). Rangel et al., J Biol Chem 280(18):17807-17814 (2005) reportthe identification and molecular characterization of a Vs-like proteinthat is the product of an unrearranged Vκ gene, which turned out to thebe identical to the cDNA sequence previously reported by Thompson etal., Immunogenetics 48:305-311 (1998). Whereas, Frances et al., EMBO J.13:5937-43 (1994) reported the identification and characterization of arearranged germline JCk that has the capacity to associate with μ heavychains at the surface of B cell precursors, thereby providing analternative to the λ5 pathway for B cell development. It has beenproposed that κ-like and λ-like pre-BCRs work in concert to promotelight chain rearrangement and ensure the maturation of B cellprogenitors. For a review, see McKeller and Martinez-Valdez Seminars inImmunology 18:4043 (2006).

The term “λ5” is used herein in the broadest sense and refers to anynative sequence or variant λ5 polypeptide, specifically including,without limitation, native sequence human and other mammalian λ5polypeptides, and variants formed by posttranslational modifications, aswell a variants of such native sequence polypeptides.

The terms “‘variant VpreB polypeptide” and “a variant of a VpreBpolypeptide” are used interchangeably, and are defined herein as apolypeptide differing from a native sequence VpreB polypeptide at one ormore amino acid positions as a result of an amino acid modification. The“variant VpreB polypeptide,” as defined herein, will be different from anative antibody λ or κ light chain sequence, or a fragment thereof. The“variant VpreB polypeptide” will preferably retain at least about 65%,or at least about 70%, or at least about 75%, or at least about 80%, orat least about 85%, or at least about 90%, or at least about 95%, or atleast about 98% sequence identity with a native sequence VpreBpolypeptide. In another preferred embodiment, the “variant VpreBpolypeptide” will be less then 95%, or less than 90%, or less then 85%,or less than 80%, or less than 75%, or less then 70%, or less than 65%,or less than 60% identical in its amino acid sequence to a nativeantibody λ or κ light chain sequence. Variant VpreB polypeptidesspecifically include, without limitation, VpreB polypeptides in whichthe non-Ig-like unique tail at the C-terminus of the VpreB sequence ispartially or completely removed. The terms “variant λ5 polypeptide” and“a variant of a λ5 polypeptide” are used interchangeably, and aredefined herein as a polypeptide differing from a native sequence λ5polypeptide at one or more amino acid positions as a result of an aminoacid modification. The “variant λ5 polypeptide,” as defined herein, willbe different from a native antibody λ or K light chain sequence, or afragment thereof. The “variant λ5 polypeptide” will preferably retain atleast about 65%, or at least about 70%, or at least about 75%, or atleast about 80%, or at least about 85%, or at least about 90%, or atleast about 95%, or at least about 98% sequence identity with a nativesequence λ5 polypeptide. In another preferred embodiment, the “‘variantλ5 polypeptide” will be less then 95%, or less than 90%, or less then85%, or less than 80%, or less than 75%, or less then 70%, or less than65%, or less than 60% identical in its amino acid sequence to a nativeantibody λ or κ light chain sequence. Variant λ5 polypeptidesspecifically include, without limitation. λ5 polypeptides in which theunique tail at the N-terminus of the λ5 sequence is partially orcompletely removed.

The term “VpreB sequence” is used herein to refer to the sequence of“VpreB,” as hereinabove defined, or a fragment thereof.

The term “λ5 sequence” is used herein to refers to the sequence of “λ5,”as hereinabove defined, or a fragment thereof.

The term “surrogate light chain sequence,” as defined herein, means anypolypeptide sequence that comprises a “VpreB sequence” and/or a “λ5sequence,” as hereinabove defined.

The terms “κ-like surrogate light chain variable domain,” “Vκ-like SLC,”and “Vκ-like” are used interchangeably, and refer to any native sequencepolypeptide that is the product of an unrearranged Vκ; gene, andvariants thereof. In one embodiment, variants of native sequence Vκ-likepolypeptides comprise a C-terminal extension (tail) relative to antibodyκ light chain sequences. In a particular embodiment, variants of nativesequence Vκ-like polypeptides retain at least part, and preferably all,of the unique C-terminal extension (tail) that distinguishes the Vκ-likepolypeptides from the corresponding antibody κ light chains. In anotherembodiment, the C-terminal tail of the variant Vκ-like polypeptide is asequence not naturally associated with the rest of the sequence. In thelatter embodiment, the difference between the C-terminal tail naturallypresent in the native Vκ-like sequence and the variant sequence mayresult from one or more amino acid alterations (substitutions,insertions, deletions, and/or additions), or the C-terminal tail may beidentical with a tail present in nature in a different Vκ-like protein.The Vκ-like polypeptides may contain amino acid alterations in regionscorresponding to one or more of antibody κ light chain CDR1, CDR2 andCDR3 sequences. In all instances, the variants can, and preferably do,include a C-terminal extension of at least four, or at least five, or atleast six, or at least seven, or at least eight, or at least nine, or atleast ten amino acids, preferably 4-100, or 4-90, or 4-80, or 4-70, or4-60, or 4-50, or 4-45, or 4-40, or 4-35, or 4-30, or 4-25, or 4-20, or4-15, or 4-10 amino acid residues relative to a native antibody κ lightchain variable region sequence. As defined herein, Vκ-like polypeptidevariant will be different from a native antibody κ or λ light chainsequence or a fragment thereof, and will preferably retain at leastabout 65%, or at least about 70%, or at least about 75%, or at leastabout 80%, or at least about 85%, or at least about 90%, or at leastabout 95%, or at least about 98% sequence identity with a nativesequence Vκ polypeptide. In another preferred embodiment, the Vκ-likepolypeptide variant will be less then 95%, or less than 90%, or lessthen 85%, or less than 80%, or less than 75%, or less then 70%, or lessthan 65%, or less than 60%, or less then 55%, or less than 50%, or lessthan 45%, or less than 40% identical in its amino acid sequence to anative antibody λ or κ light chain sequence. In other embodiments, thesequence identity is between about 40% and about 95%, or between about45% and about 90%, or between about 50% and about 85%, or between about55% and about 80%, or between about 60% and about 75%, or between about60% and about 80%, or between about 65% and about 85%, or between about65% and about 90%, or between about 65% and about 95%. In allembodiments, preferably the Vκ-like polypeptides are capable of bindingto a target.

The terms “JCκ” and “JCκ-like” are used interchangeably, and refer tonative sequence polypeptides that include a portion identical to anative sequence κ J-constant (C) region segment and a unique N-terminalextension (tail), and variants thereof. In one embodiment, variants ofnative sequence JCκ-like polypeptides comprise an N-terminal extension(tail) that distinguishes them from an antibody JC segment. In aparticular embodiment, variants of native sequence JCκ-like polypeptidesretain at least part, and preferably all, of the unique N-terminalextension (tail) that distinguishes the JCκ-like polypeptides from thecorresponding antibody κ light chain JC segments. In another embodiment,the N-terminal tail of the variant JCκ-like polypeptide is a sequencenot naturally associated with the rest of the sequence. In the latterembodiment, the difference between the N-terminal tail naturally presentin the native JCκ-like sequence and the variant sequence may result fromone or more amino acid alterations (substitutions, insertions,deletions, and/or additions), or the N-terminal tail may be identicalwith a tail present in nature in a different JCκ-like protein. Variantsof native sequence JCκ-like polypeptides may contain one or more aminoacid alterations in the part of the sequence that is identical to anative antibody κ variable domain JC sequence. In all instances, thevariants can, and preferably do, include an N-terminal extension (uniqueN-terminus) of at least four, or at least five, or at least six, or atleast seven, or at least eight, or at least nine, or at least ten aminoacids, preferably 4-100, or 4-90, or 4-80, or 4-70, or 4-60, 4-50, or4-45, or 4-40, or 4-35, or 4-30, or 4-25, or 4-20, or 4-15, or 4-10amino acid residues relative to a native antibody κ light chain JCsequence. The JCκ-like polypeptide variant, as defined herein, will bedifferent from a native antibody λ or κ light chain JC sequence, or afragment thereof, and will preferably retain at least about 65%, or atleast about 70%, or at least about 75%, or at least about 80%, or atleast about 85%, or at least about 90%, or at least about 95%, or atleast about 98% sequence identity with a native sequence JC polypeptide.In another preferred embodiment, the JCκ-like polypeptide variant willbe less then 95%, or less than 90%, or less then 85%, or less than 80%,or less than 75%, or less then 70%, or less than 65%, or less than 60%identical in its amino acid sequence to a native antibody λ or κ lightchain JC sequence. In other embodiments, the sequence identity isbetween about 40% and about 95%, or between about 45% and about 90%, orbetween about 50% and about 85%, or between about 55% and about 80%, orbetween about 60% and about 75%, or between about 60% and about 80%, orbetween about 65% and about 85%, or between about 65% and about 90%, orbetween about 65% and about 95%.

Percent amino acid sequence identity may be determined using thesequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic AcidsRes. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison programmay be downloaded from http://www.ncbi.nlm.nih.gov or otherwise obtainedfrom the National Institute of Health, Bethesda, Md. NCBI-BLAST2 usesseveral search parameters, wherein all of those search parameters areset to default values including, for example, unmask=yes, strand=all,expected occurrences=10, minimum low complexity length=15/5, multi-passe-value=0.01, constant for multi-pass=25, dropoff for final gappedalignment=25 and scoring matrix=BLOSUM62.

The terms “conjugate,” “conjugated,” and “conjugation” refer to any andall forms of covalent or non-covalent linkage, and include, withoutlimitation, direct genetic or chemical fusion, coupling through a linkeror a cross-linking agent, and non-covalent association, for examplethrough Van der Waals forces, or by using a leucine zipper.

The term “fusion” is used herein to refer to the combination of aminoacid sequences of different origin in one polypeptide chain by in-framecombination of their coding nucleotide sequences. The term “fusion”explicitly encompasses internal fusions, i.e., insertion of sequences ofdifferent origin within a polypeptide chain, in addition to fusion toone of its termini.

As used herein, the terms “peptide,” “polypeptide” and “protein” allrefer to a primary sequence of amino acids that are joined by covalent“peptide linkages.” In general, a peptide consists of a few amino acids,typically from about 2 to about 50 amino acids, and is shorter than aprotein. The term “polypeptide,” as defined herein, encompasses peptidesand proteins.

The term “amino acid” or “amino acid residue” typically refers to anamino acid having its art recognized definition such as an amino acidselected from the group consisting of: alanine (Ala); arginine (Arg);asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln);glutamic acid (Glu); glycine (Gly); histidine (His); isoleucine (Ile):leucine (Leu); lysine (Lys); methionine (Met); phenylalanine (Phe);proline (Pro); serine (Ser); threonine (Thr); tryptophan (Trp); tyrosine(Tyr); and valine (Val) although modified, synthetic, or rare aminoacids may be used as desired. Thus, modified and unusual amino acidslisted in 37 CFR 1.822(b)(4) are specifically included within thisdefinition and expressly incorporated herein by reference. Amino acidscan be subdivided into various sub-groups. Thus, amino acids can begrouped as having a nonpolar side chain (e.g., Ala, Cys, Ile, Leu, Met,Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); apositively charged side chain (e.g., Arg, His, Lys); or an unchargedpolar side chain (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr,Trp, and Tyr). Amino acids can also be grouped as small amino acids(Gly, Ala), nucleophilic amino acids (Ser, His, Thr, Cys), hydrophobicamino acids (Val, Leu, Ile, Met, Pro), aromatic amino acids (Phe, Tyr,Trp, Asp, Glu), amides (Asp, Glu), and basic amino acids (Lys, Arg).

The term “polynucleotide(s)” refers to nucleic acids such as DNAmolecules and RNA molecules and analogues thereof (e.g., DNA or RNAgenerated using nucleotide analogues or using nucleic acid chemistry).As desired, the polynucleotides may be made synthetically, e.g., usingart-recognized nucleic acid chemistry or enzymatically using, e.g., apolymerase, and, if desired, be modified. Typical modifications includemethylation, biotinylation, and other art-known modifications. Inaddition, the nucleic acid molecule can be single-stranded ordouble-stranded and, where desired, linked to a detectable moiety.

The term “variant” with respect to a reference polypeptide refers to apolypeptide that possesses at least one amino acid mutation ormodification (i.e., alteration) as compared to a native polypeptide.Variants generated by “amino acid modifications” can be produced, forexample, by substituting, deleting, inserting and/or chemicallymodifying at least one amino acid in the native amino acid sequence.

An “amino acid modification” refers to a change in the amino acidsequence of a predetermined amino acid sequence. Exemplary modificationsinclude an amino acid substitution, insertion and/or deletion.

An “amino acid modification at a specified position,” refers to thesubstitution or deletion of the specified residue, or the insertion ofat least one amino acid residue adjacent the specified residue. Byinsertion “adjacent” a specified residue is meant insertion within oneto two residues thereof. The insertion may be N-terminal or C-terminalto the specified residue.

An “amino acid substitution” refers to the replacement of at least oneexisting amino acid residue in a predetermined amino acid sequence withanother different “replacement” amino acid residue. The replacementresidue or residues may be “naturally occurring amino acid residues”(i.e. encoded by the genetic code) and selected from the groupconsisting of: alanine (Ala); arginine (Arg); asparagine (Asn); asparticacid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu);glycine (Gly); histidine (His); isoleucine (Ile): leucine (Leu); lysine(Lys); methionine (Met); phenylalanine (Phe); proline (Pro); serine(Ser); threonine (Thr); tryptophan (Trp); tyrosine (Tyr); and valine(Val). Substitution with one or more non-naturally occurring amino acidresidues is also encompassed by the definition of an amino acidsubstitution herein.

A “non-naturally occurring amino acid residue” refers to a residue,other than those naturally occurring amino acid residues listed above,which is able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Examples of non-naturally occurring amino acidresidues include norleucine, ornithine, norvaline, homoserine and otheramino acid residue analogues such as those described in Ellman et al.Meth. Enzym. 202:301 336 (1991). To generate such non-naturallyoccurring amino acid residues, the procedures of Noren et al. Science244:182 (1989) and Ellman et al., supra, can be used. Briefly, theseprocedures involve chemically activating a suppressor tRNA with anon-naturally occurring amino acid residue followed by in vitrotranscription and translation of the RNA.

An “amino acid insertion” refers to the incorporation of at least oneamino acid into a predetermined amino acid sequence. While the insertionwill usually consist of the insertion of one or two amino acid residues,the present application contemplates larger “peptide insertions”, e.g.insertion of about three to about five or even up to about ten aminoacid residues. The inserted residue(s) may be naturally occurring ornon-naturally occurring as disclosed above.

An “amino acid deletion” refers to the removal of at least one aminoacid residue from a predetermined amino acid sequence.

The term “mutagenesis” refers to, unless otherwise specified, any artrecognized technique for altering a polynucleotide or polypeptidesequence. Preferred types of mutagenesis include error prone PCRmutagenesis, saturation mutagenesis, or other site directed mutagenesis.

“Site-directed mutagenesis” is a technique standard in the art, and isconducted using a synthetic oligonucleotide primer complementary to asingle-stranded phage DNA to be mutagenized except for limitedmismatching, representing the desired mutation. Briefly, the syntheticoligonucleotide is used as a primer to direct synthesis of a strandcomplementary to the single-stranded phage DNA, and the resultingdouble-stranded DNA is transformed into a phage-supporting hostbacterium. Cultures of the transformed bacteria are plated in top agar,permitting plaque formation from single cells that harbor the phage.Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.Plaques of interest are selected by hybridizing with kinased syntheticprimer at a temperature that permits hybridization of an exact match,but at which the mismatches with the original strand are sufficient toprevent hybridization. Plaques that hybridize with the probe are thenselected, sequenced and cultured, and the DNA is recovered.

The term “neutralizing molecule” is used herein in the broadest senseand refers to any molecule that inhibits a virus from replicativelyinfecting a target cell, irrespective of the mechanism by whichneutralization is achieved, The neutralizing molecule preferably anantibody or an antibody-like molecule, as hereinabove defined,Neutralization can be achieved, for example, by inhibiting theattachment or adhesion of the virus to the cell surface, e.g., byengineering an molecule, such as an antibody or antibody-like molecule,that binds directly to, or close by, the site responsible for theattachment or adhesion of the virus. Neutralization can also be achievedby a molecule, such as an antibody or antibody-like molecule, directedto the virion surface, which results in the aggregation of virions.Neutralization can further occur by inhibition of the fusion of viraland cellular membranes following attachment of the virus to the targetcell, by inhibition of endocytosis, inhibition of progeny virus from theinfected cell, and the like. The neutralizing molecules, such asantibodies or antibody-like molecules, of the present invention are notlimited by the mechanism by which neutralization is achieved.

The term “antibody repertoire” is used herein in the broadest sense andrefers to a collection of antibodies or antibody fragments which can beused to screen for a particular property, such as binding ability,binding specificity, ability of gastrointestinal transport, stability,affinity, and the like. The term specifically includes antibodylibraries, including all forms of combinatorial libraries, such as, forexample, antibody phage display libraries, including, withoutlimitation, single-chain Fv (scFv) and Fab antibody phage displaylibraries from any source, including naïve, synthetic and semi-syntheticlibraries.

Similarly, a “repertoire of antibody-like molecules” (as hereinabovedefined) refers to a collection of such molecules which can be used toscreen for a particular property, such as binding ability, bindingspecificity, ability of gastrointestinal transport, stability, affinity,and the like. The term specifically includes surrobody libraries andlibraries of κ-like light chain constructs (as hereinabove defined),including all forms of combinatorial libraries, such as, for example,phage display libraries. Combinatorial surrobody libraries aredisclosed, for example, in Xu et al., (2008), supra.

A “phage display library” is a protein expression library that expressesa collection of cloned protein sequences as fusions with a phage coatprotein. Thus, the phrase “phage display library” refers herein to acollection of phage (e.g., filamentous phage) wherein the phage expressan external (typically heterologous) protein. The external protein isfree to interact with (bind to) other moieties with which the phage arecontacted. Each phage displaying an external protein is a “member” ofthe phage display library.

An “antibody phage display library” refers to a phage display librarythat displays antibodies or antibody fragments. The antibody libraryincludes the population of phage or a collection of vectors encodingsuch a population of phage, or cell(s) harboring such a collection ofphage or vectors. The library can be monovalent, displaying on averageone single-chain antibody or antibody fragment per phage particle, ormulti-valent, displaying, on average, two or more antibodies or antibodyfragments per viral particle. The term “antibody fragment” includes,without limitation, single-chain Fv (scFv) fragments and Fab fragments.Preferred antibody libraries comprise on average more than 10⁶, or morethan 10⁷, or more than 10⁸, or more than 10⁹ different members.

The term “filamentous phage” refers to a viral particle capable ofdisplaying a heterogenous polypeptide on its surface, and includes,without limitation, f1, fd, Pf1, and M13. The filamentous phage maycontain a selectable marker such as tetracycline (e.g., “fd-tet”).Various filamentous phage display systems are well known to those ofskill in the art (see, e.g., Zacher et al., Gene 9:127-140 (1980), Smithet al., Science 228:1315-1317 (1985); and Parmley and Smith, Gene73:305-318 (1988)).

The term “panning” is used to refer to the multiple rounds of screeningprocess in identification and isolation of phages carrying compounds,such as antibodies, with high affinity and specificity to a target.

The term “non-human animal” as used herein includes, but is not limitedto, mammals such as, for example, non-human primates, rodents (e.g.,mice and rats), and non-rodent animals, such as, for example, rabbits,pigs, sheep, goats, cows, pigs, horses and donkeys. It also includesbirds (e.g., chickens, turkeys, ducks, geese and the like). The term“non-primate animal” as used herein refers to mammals other thanprimates, including but not limited to the mammals specifically listedabove.

The phrase “functionally different antibodies,” and grammatical variantsthereof, are used to refer to antibodies that differ from each other inat least one property, including, without limitation, bindingspecificity, binding affinity, and any immunological or biologicalfunction, such as, for example, ability to neutralize a target, extentor quality of biological activity, etc.

The phrase “conserved amino acid residues” is used to refer to aminoacid residues that are identical between two or more amino acidsequences aligned with each other.

The term “epitope” as used herein, refers to a sequence of at leastabout 3 to 5, preferably at least about 5 to 10, or at least about 5 to15 amino acids, and typically not more than about 500, or about 1,000amino acids, which define a sequence that by itself, or as part of alarger sequence, binds to an antibody generated in response to suchsequence. An epitope is not limited to a polypeptide having a sequenceidentical to the portion of the parent protein from which it is derived.Indeed, viral genomes are in a state of constant change and exhibitrelatively high degrees of variability between isolates. Thus the term“epitope” encompasses sequences identical to the native sequence, aswell as modifications, such as deletions, substitutions and/orinsertions to the native sequence. Generally, such modifications areconservative in nature but non-conservative modifications are alsocontemplated. The term specifically includes “mimotopes,” i.e. sequencesthat do not identify a continuous linear native sequence or do notnecessarily occur in a native protein, but functionally mimic an epitopeon a native protein. The term “epitope” specifically includes linear andconformational epitopes.

B. General Techniques

Techniques for performing the methods of the present invention are wellknown in the art and described in standard laboratory textbooks,including, for example, Ausubel et al., Current Protocols of MolecularBiology, John Wiley and Sons (1997); Molecular Cloning: A LaboratoryManual, Third Edition, J. Sambrook and D. W. Russell, eds., Cold SpringHarbor, N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; AntibodyPhage Display: Methods and Protocols, P. M. O'Brian and R. Aitken, eds.,Humana Press, In: Methods in Molecular Biology, Vol. 178; Phage Display:A Laboratory Manual, C. F. Barbas III et al. eds., Cold Spring Harbor,N.Y., USA, Cold Spring Harbor Laboratory Press, 2001; and Antibodies, G.Subramanian, ed., Kluwer Academic, 2004. Mutagenesis can, for example,be performed using site-directed mutagenesis (Kunkel et al., Proc. Natl.Acad. Sci. USA 82:488-492 (1985)).

In one aspect, the viral antigen neutralizing molecules of the presentinvention are antibodies, which are typically selected using antibodylibraries. In the following description, the invention is illustratedwith reference to certain types of antibody libraries, but the inventionis not limited to the use of any particular type of antibody library.Recombinant monoclonal antibody libraries can be based on immunefragments or naïve fragments. Antibodies from immune antibody librariesare typically constructed with V_(H) and V_(L) gene pools that arecloned from source B cells into an appropriate vector for expression toproduce a random combinatorial library, which can subsequently beselected for and/or screened. Other types of libraries may be comprisedof antibody fragments from a source of genes that is not explicitlybiased for clones that bind to an antigen. Thus, naïve antibodylibraries derive from natural, unimmunized, rearranged V genes.Synthetic antibody libraries are constructed entirely by in vitromethods, introducing areas of complete or tailored degeneracy into theCDRs of one or more V genes. Semi-synthetic libraries combine naturaland synthetic diversity, and are often created to increase naturaldiversity while maintaining a desired level of functional diversity.Thus, such libraries can, for example, be created by shuffling naturalCDR regions (Soderlind et al., Nat. Biotechnol. 18:852-856 (2000)), orby combining naturally rearranged CDR sequences from human B cells withsynthetic CDR1 and CDR2 diversity (Hoet et al., Nat. Biotechnol.23:455-38 (2005)). The present invention encompasses the use of naïve,synthetic and semi-synthetic antibody libraries, or any combinationthereof.

Similarly, the methods of the present invention are not limited by anyparticular technology used for the display of antibodies. Although theinvention is illustrated with reference to phage display, antibodies ofthe present invention can also be identified by other display andenrichment technologies. Antibody fragments have been displayed on thesurface of filamentous phage that encode the antibody genes (Hoogenboomand Winter J. Mol. Biol., 222:381 388 (1992); McCafferty et al., Nature348(6301):552 554 (1990); Griffiths et al. EMBO J, 13(14):3245-3260(1994)). For a review of techniques for selecting and screening antibodylibraries see, e.g., Hoogenboom, Nature Biotechnol. 23(9):1105-1116(2005). In addition, there are systems known in the art for display ofheterologous proteins and fragments thereof on the surface ofEscherichia coli (Agterberg et al., Gene 88:37-45 (1990); Charbit etal., Gene 70:181-189 (1988); Francisco et al., Proc. Natl. Acad. Sci.USA 89:2713-2717 (1992)), and yeast, such as Saccharomyces cerevisiae(Boder and Wittrup, Nat. Biotechnol. 15:553-557 (1997); Kieke et al.,Protein Eng. 10:1303-1310 (1997)). Other known display techniquesinclude ribosome or mRNA display (Mattheakis et al., Proc. Natl. Acad.Sci. USA 91:9022-9026 (1994); Hanes and Pluckthun, Proc. Natl. Acad.Sci. USA 94:4937-4942 (1997)), DNA display (Yonezawa et al., Nucl. AcidRes. 31(19):e118 (2003)); microbial cell display, such as bacterialdisplay (Georgiou et al., Nature Biotech. 15:29-34 (1997)), display onmammalian cells, spore display (Isticato et al., J. Bacteriol.183:6294-6301 (2001); Cheng et al., Appl. Environ. Microbiol.71:3337-3341 (2005) and co-pending provisional application Ser. No.60/865,574, filed Nov. 13, 2006), viral display, such as retroviraldisplay (Urban et al., Nucleic Acids Res. 33:e35 (2005), display basedon protein-DNA linkage (Odegrip et al., Proc. Acad. Natl. Sci. USA101:2806-2810 (2004); Reiersen et al., Nucleic Acids Res. 33:e10(2005)), and microbead display (Sepp et al., FEBS Lett. 532:455-458(2002)).

C. Detailed Description of Preferred Embodiments

In one aspect, the present invention concerns the selection, productionand use of monoclonal antibodies and antibody-like moleculesneutralizing more than one subtype and/or more than one isolate of aninfluenza A virus, binding to a hemagglutinin (HA) antigen of the virus,but not inhibiting hemagglutination.

The virions of influenza A virus contain 8 segments of linearnegative-sense single stranded RNA. The total genome length is 13600nucleotides, and the eight segments are 2350 nucleotides; 2350nucleotides; of 2250 nucleotides; 1780 nucleotides; 1575 nucleotides;1420 nucleotides; 1050 nucleotides; and 900 nucleotides, respectively,in length. Host specificity and attenuation of influenza A virus havebeen attributed to viral hemagglutinin (H, HA), nucleoprotein (NP),matrix (M), and non-structural (NS) genes individually or incombinations of viral genes (see, e.g., Rogers et al., Virology127:361-373 (1983); Scholtissek et al., Virology 147:287-294 (1985);Snyder et al., J. Clin. Microbiol. 24:467-469 (1986); Tian et al., J.Virol. 53:771-775 (1985); Treanor et al., Virology 171:1-9 (1989).

Nucleotide and amino acid sequences of influenza A viruses and theirsurface proteins, including hemagglutinins and neuraminidase proteins,are available from GenBank and other sequence databases, such as, forexample, the Influenza Sequence Database maintained by the TheoreticalBiology and Biophysics Group of Los Alamos National Laboratory. Theamino acid sequences of 15 known H subtypes of the influenza A virushemagglutinin (H1-H15) are shown in U.S. Application Publication No.20080014205, published on Jan. 17, 2008, incorporated herein byreference in its entirety. An additional influenza A virus hemagglutininsubtype (H16) was isolated recently from black-headed gulls in Sweden,and reported by Fouchier et al., J. Virol. 79(5):2814-22 (2005). A largevariety of strains of each H subtype are also known. For example, thesequence of the HA protein designated H5 A/Hong Kong/156/97 wasdetermined from an influenza A H5N1 virus isolated from a human in HongKong in May 1997, and is shown in comparison with sequences of severaladditional strains obtained from other related H5N1 isolates in Suarezet al., J. Virol. 72:6678-6688 (1998).

The structure of the catalytic and antigenic sites of influenza virusneuraminidase have been published by Colman et al., Nature 303:41-4(1983), and neuraminidase sequences are available from GenBank and othersequence databases.

It has been known that virus-specific antibodies resulting from theimmune response of infected individuals typically neutralize the virusvia interaction with the viral hemagglutinin (Ada et al., Curr. Top.Microbiol. Immunol. 128:1-54 (1986); Couch et al., Annu. Rev. Micobiol.37:529-549 (1983)). The three-dimensional structures of influenza virushemagglutinins and crystal structures of complexes between influenzavirus hemagglutinins and neutralizing antibodies have also beendetermined and published, see, e.g., Wilson et al., Nature 289:366-73(1981); Ruigrok et al., J. Gen. Virol. 69 (Pt 11):2785-95 (1988);Wrigley et al., Virology 131(2):308-14 (1983); Daniels et al., EMBO J.6:1459-1465 (1987); and Bizebard et al., Nature 376:92-94 (2002).

According to the present invention, antibodies with the desiredproperties are identified from one or more antibody libraries, which cancome from a variety of sources and can be of different types.

Comprehensive Human Influenza Antibody Libraries

Comprehensive human influenza antibody libraries can be created fromantibodies obtained from convalescent patients of various priorinfluenza, seasonal outbreaks epidemics, and pandemics, including the1968 Hong Kong flu (H3N2), the 1957 Asian flu (H2N2), the 1918 Spanishflu (H1N1), and the 2004/2005 Avian flu (H5N1). For example, see U.S.Application Publication No. 20080014205, published on Jan. 17, 2008,incorporated herein by reference in its entirety. In order to preparesuch libraries, blood or bone marrow samples are collected fromindividuals known or suspected to have been infected with an influenzavirus. Peripheral blood samples, especially from geographically distantsources, may need to be stabilized prior to transportation and use. Kitsfor this purpose are well known and commercially available, such as, forexample, BD Vacutainer® CPT™ cell preparation tubes can be used forcentrifugal purification of lymphocytes, and guanidium, Trizol, orRNAlater used to stabilize the samples. Upon receipt of the stabilizedlymphocytes or whole bone marrow, RT-PCR is performed to rescue heavyand light chain repertoires, using immunoglobulin oligo primers known inthe art. The PCR repertoire products are combined with linker oligos togenerate scFv libraries to clone directly in frame with m13 pIIIprotein, following procedures known in the art.

In a typical protocol, antibodies in the human sera can be detected bywell known serological assays, including, for example, by the well-knownhemagglutinin inhibition (HAI) assay (Kendal, A. P., M. S. Pereira, andJ. J. Skehel. 1982. Concepts and procedures for laboratory-basedinfluenza surveillance. U.S. Department of Health and Human Services,Public Health Service, Centers for Disease Control, Atlanta, Ga.), orthe microneutralization assay (Harmon et al., J. Clin. Microbiol.26:333-337 (1988)). This detection step might not be necessary if theserum sample has already been confirmed to contain influenzaneutralizing antibodies. Lymphocytes from whole blood or those presentin bone marrow are next processed by methods known in the art. Whole RNAis extracted by Tri BD reagent (Sigma) from fresh or RNAlater stabilizedtissue. Subsequently, the isolated donor total RNA is further purifiedto mRNA using Oligotex purification (Qiagen). Next first strand cDNAsynthesis, is generated by using random nonamer oligonucleotides and oroligo (dT)₁₈ primers according to the protocol of AccuScript reversetranscriptase (Stratagene). Briefly, 100 ng mRNA, 0.5 mM dNTPs and 300ng random nonamers and or 500 ng oligo (dT)₁₈ primers in Accuscript RTbuffer (Stratagene) are incubated at 65° C. for 5 min, followed by rapidcooling to 4° C. Then, 100 mM DTT, Accuscript RT, and RNAse Block areadded to each reaction and incubated at 42° C. for 1 h, and the reversetranscriptase is inactivated by heating at 70° C. for 15 minutes. ThecDNA obtained can be used as a template for RT-PCR amplification of theantibody heavy and light chain V genes, which can then be cloned into avector, or, if phage display library is intended, into a phagemidvector. This procedure generates a repertoire of antibody heavy andlight chain variable region clones (V_(H) and V_(L) libraries), whichcan be kept separate or combined for screening purposes.

Immunoglobulin repertoires from peripheral lymphocytes of survivors ofearlier epidemics and pandemics, such as the 1918 Spanish Flu, can beretrieved, stabilized, and rescued in a manner similar to that describedabove. For additional H1 and H3 libraries repertoires can be recoveredfrom properly timed vaccinated locally-sourced donors. As an additionaloption commercially available bone marrow total RNA or mRNA can bepurchased from commercial sources to produce libraries suitable for H1and H3, and depending upon the background of donor also suitable for H2antibody screening.

Synthetic Human-Like Repertoire

In the methods of the present invention, the synthetic human antibodyrepertoire can be represented by a synthetic antibody library, which canbe made by methods known in the art or obtained from commercial sources.Thus, for example, a fully synthetic human repertoire is described inU.S. patent application Ser. No. 11/864,525 filed on Sep. 28, 2007, theentire disclosure of which is hereby expressly incorporated byreference. In brief, this patent application describes libraries ofimmunoglobulins in which predetermined amino acids have beencombinatorially introduced into one or more complementarity-determiningregions of the immunoglobulin of interest. Additionally, for example, auniversal immunoglobulin library, including subsets of such library, aredescribed in U.S. Patent Application Publication No. 20030228302published on Dec. 11, 2003, the entire disclosure of which is herebyexpressly incorporated by reference.

Specific sublibraries of antibody heavy and light chains with variousmutations can be combined to provide the framework constructs for theantibodies of the present invention, which is followed by introducingdiversity in the CDRs of both heavy and light chains. This diversity canbe achieved by methods known in the art, such as, for example, by Kunkelmutagenesis, and can be repeated several times in order to furtherincrease diversity. Thus, for example, diversity into the heavy andlight chain CDR1 and CD2 regions, separately or simultaneously, can beintroduced by multiple rounds of Kunkel mutagenesis. If necessary, thevarious Kunkel clones can be segregated by CDR lengths and/or cloneslacking diversity in a targeted CDR (e.g., CDR1 or CDR3) can be removed,e.g., by digestion with template-specific restriction enzymes. Uponcompletion of these steps, the size of the library should exceed about10⁹ members, but libraries with lesser members are also useful.

In a specific embodiment, both immunized antibody libraries andsynthetic antibody libraries are used for identifying the neutralizingantibodies of the present invention. The two types of libraries arefundamentally different. The synthetic antibody libraries aresynthesized collections of human antibodies with the predicted abilityto bind antigens, while an immunized repertoire will contain sequencesto specifically recognize avian H5 hemagglutinin, and/or H1, H2, or H3hemagglutinin, as the case may be. Thus, the immunized repertoires aretheoretically optimized to recognize critical components of targetedinfluenza subtype(s). As a result these differences the two methodsproduce a different set of antibodies and thus provide a more efficientapproach for identifying the desired neutralizing antibodies.

Hyperimmunized Non-Human Primate Antibody Libraries

In this method, an antibody library is rescued from hyperimmunizednon-human primates, such as, for example, macaque or baboons.Specifically, non-human primates are immunized with various subtypes ofthe influenza A virus or with various hemagglutinin (H) proteins.Animals developing titers of antibody recognizing the influenza A virussubtype or hemagglutinin they were immunized with are sacrificed andtheir spleens harvested. Blood or bone marrow of the immunized animalsis collected, and antibodies produced are collected and amplified asdescribed above for the comprehensive influenza antibody libraries.

Strategies for Isolating Neutralizing Antibodies of the Invention

Regardless of the type of antibody library or libraries used, antibodieswith dual specificities, such as, for example, showing reactivity withtwo different influenza A subtypes and/or with two strains (isolates) ofthe same subtype, and/or with human and non-human isolates, can bediscovered and optimized through controlled cross-reactive selectionand/or directed combinatorial and/or mutagenic engineering.

In a typical enrichment scheme, illustrated in FIG. 1, a libraryincluding antibodies showing cross-reactivity to two targets, designatedas targets A and B, are subjected to multiple rounds of enrichment (seeU.S. Application Publication No. 20080014205, published on Jan. 17,2008, incorporated herein by reference in its entirety). If enrichmentis based on reactivity with target A, each round of enrichment willincrease the reactive strength of the pool towards target A. Similarly,if enrichment is based on reactivity with target B, each round ofenrichment will increase the reactive strength of the pool towardstarget B. Although this approach refers to panning, which is theselection method used when screening phage display libraries (seebelow), the approach is equally applicable to any type of librarydiscussed above, other otherwise known in the art, and to any type ofdisplay technique. Targets A and B include any targets to whichantibodies bind, including but not limited to various isolates, typesand sub-types of influenza viruses.

Since the goal of the present invention is to identify neutralizingantibodies with multiple specificities, a cross-reactive discoveryselection scheme has been developed. In the interest of simplicity, thisscheme is illustrated in FIG. 2 showing the selection of antibodies withdual specificities. In this case, an antibody library includingantibodies showing reactivity with two targets, targets A and B, isfirst selected for reactivity with one of the targets, e.g., target A,followed by selection for reactivity with the other target, e.g., targetB. Each successive selection round reinforces the reactive strength ofthe resulting pool towards both targets. (see also U.S. ApplicationPublication No. 20080014205, published on Jan. 17, 2008, incorporatedherein by reference in its entirety). Accordingly, this method isparticularly useful for identifying antibodies with dual specificity. Ofcourse, the method can be extended to identifying antibodies showingreactivity towards further targets, by including additional rounds ofenrichment towards the additional target(s). Again, if the libraryscreened is a phage display library, selection is performed bycross-reactive panning, but other libraries and other selection methodscan also be used.

A combination of the two methods discussed above includes two separateenrichment rounds for reactivity towards target A and target B,respectively, recombining the two pools obtained, and subsequentcross-reactive selection rounds, as described above (see U.S.Application Publication No. 20080014205, published on Jan. 17, 2008,incorporated herein by reference in its entirety). This approach isillustrated in FIG. 3. Just as in the pure cross-reactive selection,each round of selection of the recombined library increases the reactivestrength of the resulting pool towards both targets.

In a further embodiment, illustrated in FIG. 4, first a clone showingstrong reactivity with a target A, and having detectablecross-reactivity with target B is identified. Based on this clone, amutagenic library is prepared, which is then selected, in alternatingrounds, for reactivity with target B and target A respectively. Thisscheme will result in antibodies that maintain strong reactivity withtarget A, and have increased reactivity with target B (see U.S.Application Publication No. 20080014205, published on Jan. 17, 2008,incorporated herein by reference in its entirety). Just as before,selection is performed by panning, if the libraries screened are phagedisplay libraries, but other libraries, other display techniques, andother selection methods can also be used, following the same strategy.

As discussed above, targets A and B can, for example, be two differentsubtypes of the influenza A virus, two different strains (isolates) ofthe same influenza A virus, subtypes or isolates from two differentspecies, where one species is preferably human. Thus, for example,target A may be an isolate of the 2004 Vietnam isolate of the H5N1virus, and target B may be a 1997 Hong Kong isolate of the H5N1 virus.It is emphasized that these examples are merely illustrative, andantibodies with dual and multiple specificities to any two or multipletargets can be identified, selected and optimized in an analogousmanner.

Alternatively, if an antibody library such as the UAL that allowssegregation of discrete frameworks and CDR lengths is used to find anantibody to target A, then an antigen B could be screened for and thelibrary could be restricted to a diverse collection of similarparameters. Once an antibody to antigen B is found then chimeric ormutagenic antibodies based upon the respective A and B antibodies couldbe used to engineer a dual specific collection.

Phage Display

In a particular embodiment, the present invention utilizes phage displayantibody libraries to functionally discover neutralizing monoclonalantibodies with multiple (including dual) specificities. Such antibodiescan, for example, be monoclonal antibodies capable of neutralizing morethan one influenza A virus subtype, including the H5, H7 and/or H9subtypes, such as the H5 and H1; H5 and H2; H5 and H3; H5, H1, and H2;H5, H1, and H3; H5, H2 and H3; H1, H2 and H3, etc., subtypes, and/ormore than one strain (isolate) of the same subtype.

To generate a phage antibody library, a cDNA library obtained from anysource, including the libraries discussed above, is cloned into aphagemid vector.

Thus, for example, the collection of antibody heavy and light chainrepertoires rescued from lymphocytes or bone marrow by RT-PCR asdescribed above, is reassembled as a scFv library fused to m13 pIIIprotein. The combinatorial library will contain about more than 10⁶, ormore than 10⁷, or more than 10⁸, or more than 10⁹ different members,more than 10⁷ different members or above being preferred. For qualitycontrol random clones are sequenced to assess overall repertoirecomplexity.

Similarly, following the initial PCR rescue of heavy and light chainvariable regions from a naïve or immunized human, or hyperimmunizednonhuman primate antibody library, the PCR products are combined withlinker oligos to generate scFv libraries to clone directly in frame withM13 pIII coat protein. The library will contain about more than 10⁶, ormore than 10⁷, or more than 10⁸, or more than 10⁹ different members,more than 10⁷ different members or above being preferred. As a qualitycontrol step, random clones are sequenced in order to assess overallrepertoire size and complexity.

Antibody phage display libraries may contain antibodies in variousformats, such as in a single-chain Fv (scFv) or Fab format. For reviewsee, e.g., Hoogenboom, Methods Mol. Biol. 178:1-37 (2002).

Screening

Screening methods for identifying antibodies with the desiredneutralizing properties have been described above. Reactivity can beassessed based on direct binding to the desired hemagglutinin proteins.

Hemagglutinin (HA) Protein Production

Hemagglutinin (HA) proteins can be produced by recombinant DNAtechnology. In this method, HA genes are cloned into an appropriatevector, preferably a baculovirus expression vector for expression inbaculovirus-infected insect cells, such as Spodoptera frugiperda (Sf9)cells.

The nucleic acid coding for the HA protein is inserted into abaculovirus expression vector, such as Bac-to-Bac (Invitrogen), with orwithout a C-terminal epitope tag, such as a poly-his (hexahistidinetag). A poly-his tag provides for easy purification by nickel chelatechromatography.

In general the cloning involves making reference cDNAs by assembly PCRfrom individually synthesized oligos. Corresponding isolate variant HAproteins are made by either substituting appropriate mutant oligos intoadditional assembly PCRs or by mutagenesis techniques, such as by Kunkelmutagenesis. Two clusters of HA protein sequences exist for H5, the 1997and 2004 subtype isolates. Therefore, a single reference protein is madefor each cluster. Similarly, reference proteins are generated for 1918Spanish flu (H1), 1958 Asian Flu (H2), 1968 Hong Kong Flu (H3), andcurrent H1, H2, H3 isolates.

Recombinant baculovirus is generated by transfecting the above Bacmidinto Sf9 cells (ATCC CRL 1711) using lipofectin (commercially availablefrom Gibco-BRL). After 4-5 days of incubation at 28° C., the releasedviruses are harvested and used for further amplifications. Viralinfection and protein expression are performed as described by O'Reilleyet al., Baculovirus Expression Vectors: A Laboratory Manual (Oxford:Oxford University Press, 1994).

Expressed poly-His-tagged HA polypeptides can then be purified, forexample, by Ni²⁺-chelate affinity chromatography as follows.Supernatants are collected from recombinant virus-infected Sf9 cells asdescribed by Rupert et al., Nature 362:175-179 (1993). A Ni²⁺-NTAagarose column (commercially available from Qiagen) is prepared with abed volume of 5 mL, washed with 25 mL of water, and equilibrated with 25mL of loading buffer. The filtered cell extract is loaded onto thecolumn at 0.5 mL per minute. The column is washed to baseline A₂₈₀ withloading buffer, at which point fraction collection is started. Next, thecolumn is washed with a secondary wash buffer (50 mM phosphate; 300 mMNaCl, 10% glycerol, pH 6.0), which elutes non-specifically boundprotein. After reaching A₂₈₀ baseline again, the column is developedwith a 0 to 500 mM imidazole gradient in the secondary wash buffer.One-mL fractions are collected and analyzed by SDS-PAGE and silverstaining or Western blot with Ni²⁺-NTA-conjugated to alkalinephosphatase (Qiagen). Fractions containing the eluted His₁₀-tagged HApolypeptide are pooled and dialyzed against loading buffer.

Alternatively, purification of an IgG-tagged (or Fc-tagged) HApolypeptide can be performed using known chromatography techniques,including, for instance, Protein A or protein G column chromatography.

As an alternative to using Sf9 cells HA proteins can also be produced inother recombinant host cells, prokaryote, yeast, or higher eukaryotecells. Suitable prokaryotes include but are not limited to eubacteria,such as Gram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325); and K5 772 (ATCC53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for vectorscontaining nucleic acid encoding an HA polypeptide. Saccharomycescerevisiae is a commonly used lower eukaryotic host microorganism.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe (Beachand Nurse, Nature 290: 140 (1981); EP 139,383 published 2 May 1985);Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol. 737 (1983)), K.fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van denBerg et al., Bio/Technology 8:135 (1990)), K. thermotolerans, and K.marxianus, yarrowia (EP 402,226); Pichia pastoris (EP 183,070;Sreekrishna et al., J. Basic Microbiol. 28:265-278 (1988)); Candida,Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc.Natl. Acad. Sci. USA 76:5259-5263 (1979)); Schwanniomyces such asSchwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990); andfilamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium(WO 91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.nidulans (Ballance et al., Biochem. Biophys. Res. Commun. 112:284-289(1983); Tilburn et al., Gene 26:205-221 (1983); Yelton et al., Proc.Natl. Acad. Sci. USA 81:1470-1474 (1984)) and A. niger Kelly and Hynes,EMBO J. 4:475-479 (1985). Methylotropic yeasts are suitable herein andinclude, but are not limited to, yeast capable of growth on methanolselected from the genera consisting of Hansenula, Candida, Kloeckera,Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specificspecies that are exemplary of this class of yeasts may be found in C.Anthony, The Biochemistry of Methylotrophs 269 (1982).

Suitable host cells for the expression of HA proteins include cells ofmulticellular organisms. Examples of invertebrate cells include theabove-mentioned insect cells such as Drosophila S2 and Spodoptera Sf9,as well as plant cells. Examples of useful mammalian host cell linesinclude Chinese hamster ovary (CHO) and COS cells. More specificexamples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCCCRL 1651); human embryonic kidney line (HEK 293 or HEK 293 cellssubcloned for growth in suspension culture (Graham et al., J. Gen Virol.36:59 (1977)); Chinese hamster ovary cells/−DHFR (CHO, Urlaub andChasin, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells(TM4, Mather, Biol. Reprod. 23:243-251 (1980)); human lung cells (WI 38,ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammarytumor (MMT 060562, ATCC CCL51). The selection of the appropriate hostcell is deemed to be within the skill in the art.

Hemagglutinin (HA) Protein Panning

HA protein is immobilized on to the surface of microtiter wells ormagnetic beads to pan the described above libraries. In a particularembodiment, each library is allowed to bind the H5 protein at 4 degreesfor two hours and then washed extensively with cold PBS, before elutingHA specific binding clones with 0.2M glycine-HC1 buffer (pH2.5). Therecovered phage is pH neutralized and amplified by infecting asusceptible host E. coli. Subsequently, phagemid production can beinduced to repeat the enrichment of positive clones and subsequentclones isolation for triage. Upon sufficient enrichment the entire poolis transferred by infection into a non amber suppressor E. coli strainsuch as HB2151 to express soluble scFv proteins. Alternatively thepool(s) could be subcloned into a monomeric scFv expression vector, suchas pBAD, and recombinant soluble scFv proteins are expressed for invitro analysis and characterization, as described below.

Characterization

H5 clones are first tested for binding affinity to an H5 proteinproduced as described above. In a particular example, binding is testedto a 2004 H5 protein (Refseq AAS65618, Isolate;A/Thailand/2(SP-33)/2004(H5N1)), and in parallel test to a 1997 H5protein (Refseq AAF74331, Isolate; A/Hong Kong/486/97(H5N1)), but otherisolates can also be used alone or in any combination. The positiveclones obtained with the 2004 and the 1997 H5 proteins will fall intotwo broad categories: 2004 selective and 2004/1997 nonselective. Thetypical functional test for neutralization involves hemagglutinationinhibition assays using whole virus binding to red blood cells. Due tosafety concerns, alternative hemagglutination assays with recombinantprotein and red blood cells are preferred. In order to eliminate theneed for whole blood, the hemagglutinin binding inhibition assay can bepreformed on airway epithelial cells. The binding assay can be performedin any configuration, including, without limitation, any flow cytometricor cell ELISA (cELISA) based assays. Using cELISA is advantageous inthat it obviates the use of expensive flow cytometry equipment and canprovide for more automated clonal assessment and greater datacollection. On the other hand, flow cytometry may provide greatersensitivity, consistency, and speed.

H1 clones can be tested for binding to any H1 proteins, includingbinding to the current 2004 H1 and, in parallel, for binding to 1918 and1976 proteins. The positive clones will fall into two broad categories:2004 selective and 2004 nonselective. Once again it is critical to testfor neutralization, using methodologies similar to those describedabove.

Other HA proteins, such as H2, H3, H5, H6, H7, H8 and H9, can becharacterized in an analogous manner.

In one aspect, the antibodies of the present invention have a bindingaffinity for an H2, H3, H5, H6, H7, H8, or H9 HA containing influenzavirus or an H1 HA containing influenza virus, such as, for example,H1/H3, H1/H5, etc. Binding affinities of the antibodies of the presentinvention can be determined by methods known to those of skill in theart, for example by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980). In one embodiment, the binding affinity of theantibody is from about 1×10⁻⁷ to about 1×10⁻¹³ M, from about 1×10⁻⁸ toabout 1×10⁻¹² M, or from about 1×10⁻⁹ to about 1×10⁻¹¹ M. In otherembodiments, the binding affinity of the antibody is about 1×10⁻⁷ M,about 1×10⁻⁸ M, about 1×10⁻⁹ M, about 1×10⁻¹⁰ M, about 1×10⁻¹¹ M, about1×10⁻¹² M, or about 1×10⁻¹³ M. For example, an antibody of the presentinvention demonstrated a binding affinity of 13 μM for an H5 HA(Vietnam/1203/04) containing influenza virus (see antibodies fromsurvivor 2 in the Example below). Another antibody demonstrated abinding affinity of single digit nM for an H5 HA (Vietnam/1203/04)containing influenza virus (see antibodies from survivor 5 in theExample below).

Optimization

For the efficient management of influenza epidemics and pandemics,including a potential pandemic associated with human infections causedby an avian (H5) virus, antibodies that effectively neutralize currentisolates of the H proteins, such as the H5 protein, as well as futuremutations, are needed. In order to achieve this goal, diverse H (e.g.,H5) neutralizing clones need to be identified that bind all knownisolates of the targeted hemagglutinin subtype(s).

If desired, cross-reactivity can be further improved by methods known inthe art, such as, for example, by Look Through Mutagenesis (LTM), asdescribed in US. Patent Application Publication No. 20050136428,published Jun. 23, 2005, the entire disclosure of which is herebyexpressly incorporated by reference.

Look-through mutagenesis (LTM) is a multidimensional mutagenesis methodthat simultaneously assesses and optimizes combinatorial mutations ofselected amino acids. The process focuses on a precise distributionwithin one or more complementarity determining region (CDR) domains andexplores the synergistic contribution of amino acid side-chainchemistry. LTM generates a positional series of single mutations withina CDR where each wild type residue is systematically substituted by oneof a number of selected amino acids. Mutated CDRs are combined togenerate combinatorial single-chain variable fragment (scFv) librariesof increasing complexity and size without becoming prohibitive to thequantitative display of all variants. After positive selection, cloneswith improved properties are sequenced, and those beneficial mutationsare mapped. To identify synergistic mutations for improved HA bindingproperties, combinatorial libraries (combinatorial beneficial mutations,CBMs) expressing all beneficial permutations can be produced by mixedDNA probes, positively selected, and analyzed to identify a panel ofoptimized scFv candidates. The procedure can be performed in a similarmanner with Fv and other antibody libraries.

Mutagenesis can also be performed by walk-through mutagenesis (WTM), asdescribed above.

Another useful mutagenic method to intentionally design cross-reactivityof the antibodies herein with more than one influenza A subtype and/ormore than one isolate of the same subtype, is referred herein as“destinational” mutagenesis. Destinational mutagenesis can be used torationally engineer a collection of antibodies based upon one or moreantibody clones, preferably of differing reactivities. In the context ofthe present invention, destinational mutagenesis is used to encodesingle or multiple residues defined by analogous positions on likesequences such as those in the individual CDRs of antibodies. In thiscase, these collections are generated using oligo degeneracy to capturethe range of residues found in the comparable positions. It is expectedthat within this collection a continuum of specificities will existbetween or even beyond those of the parental clones. The objective ofdestinational mutagenesis is to generate diverse multifunctionalantibody collections, or libraries, between two or more discreteentities or collections. In the case of influenza this method can beutilized to use two antibodies that recognize two distinct epitopes,isolates, or subtypes and morph both functional qualities into a singleantibody. As an example, a first influenza A antibody can be specific toa Vietnam isolate of the H5 subtype and a second antibody is specific toa Thailand or Turkish isolate of the H5 subtype of the influenza Avirus. To create a destinational mutagenesis library, the CDR sequencesfor both antibodies are first attained and aligned. Next all positionsof conserved identity are fixed with a single codon to the matchedresidue. At non-conserved positions a degenerate codon is incorporatedto encode both residues. In some instances the degenerate codon willonly encode the two parental residues at this position. However, in someinstances additional co-products are produced. The level of co-productproduction can be dialed in to force co-product production or eliminatethis production dependent upon size limits or goals.

Thus, for example, if the first position of the two antibodiesrespectively are threonine and alanine, the degenerate codon with A/G-C-in the first two positions would only encode threonine or alanine,irrespective of the base in the third position. If, for example, thenext position residues are lysine and arginine the degenerate codonA-A/G-A/G will only encode lysine or arginine. However, if thedegenerate codon A/C-A/G-A/G/C/T were used then asparagine, histidine,glutamine, and serine coproducts will be generated as well.

As a convenience it is simpler to use only antibodies with matched CDRlengths. One way to force this is to screen a size restricted libraryfor the second antigen, based on the CDR length and potentially evenframework restrictions imparted by the initially discovered antibody. Itis noted, however, that using CDRs of equal length is only a convenienceand not a requirement. It is easy to see that, while this method will beuseful to create large functionally diverse libraries of influenza Avirus neutralizing antibodies, its applicability is much broader. Thismutagenesis technique can be used to produce functionally diverselibraries or collections of any antibody (see U.S. ApplicationPublication No. 20080014205, published on Jan. 17, 2008 and incorporatedherein by reference in its entirety). Thus, FIG. 5 is included herein toillustrate the use of the destinational mutagenesis method using CDRs ofa TNF-α antibody and a CD11a antibody as the parental sequencesmutagenized.

Other exemplary mutagenesis methods include targeted random mutagenesis,saturation mutagenesis and error prone PCR.

Targeted random mutagenesis (Matteuchi and Heyneker, Nucleic AcidsResearch 11: 3113-3121 (1983)) using ambiguously synthesizedoligonucleotides is a technique that generates an intended codon as wellas all possible codons at specific ratios, with respect to each other,at designated positions. Ambiguously synthesized oligonucleotides resultin the reduced accuracy of nucleotide addition by the specific additionof non “wild type” bases at designated positions, or codons. This istypically performed by fixing the ratios of wild type and non wild typebases in the oligonucleotide synthesizer and designating the mixture ofthe two reagents at the time of synthesis.

Saturation mutagenesis (Hayashi et al., Biotechniques 17:310-315 (1994))is a technique in which all 20 amino acids are substituted in aparticular position in a protein and clones corresponding to eachvariant are assayed for a particular phenotype. (See, also U.S. Pat.Nos. 6,171,820; 6,358,709 and 6,361,974.)

Error prone PCR (Leung et al., Technique 1:11-15 (1989); Cadwell andJoyce, PCR Method Applic. 2:28-33 (1992)) is a modified polymerase chainreaction (PCR) technique introducing random point mutations into clonedgenes. The resulting PCR products can be cloned to produce random mutantlibraries or transcribed directly if a T7 promoter is incorporatedwithin the appropriate PCR primer.

Other mutagenesis techniques are also well known and described, forexample, in In Vitro Mutagenesis Protocols, J. Braman, Ed., HumanaPress, 2001.

In the present case, one of the main goals is to engineer an antibody(or antibodies) to effectively treat current H5 (or H7 or H9) isolatesas well as future mutations. To engineer an antibody with tolerancescapable of recognizing mutations in new isolates H5 neutralizing clonesthat bind a variety of H5 isolates, including, for example, both recent2004 isolates and previous 1997 isolates are to be identified. It isexpected that if a clone is selected on a 2004 isolate it willbind/neutralize a 1997 isolate to a lesser degree. In this case the goalis to improve 1997 recognition dramatically within the context ofimproving (or at least maintaining) 2004 isolate binding. Therefore,selection is first done for improvements on 1997 reference proteinfollowed by selection on the 2004 protein. Doing so provides a greaterselective pressure on the new strain, while maintaining pressure on thesecond parameter.

Optimization can be based on any of the libraries discussed above, orany other types of libraries known in the art, alone or in anycombination. In a particular embodiment, optimization can begin byscreening three types of LTM libraries; triple mutagenized light chainlibrary, triple mutagenized heavy chain library, and hextuplemutagenized (light+heavy chain) library. H5 is panned essentially asdescribed above, although minor modifications might be desirable. Forexample, prior to glycine-HCl elution one can select for improvedbinding by increasing washing stringencies at each round by either orboth of the following methods: extensive washing at RT or 37 degrees, orprolonged incubation in presence of excess soluble parent scFv. Theseselection modifications should improve off-rate kinetics in theresulting clones. After 3-4 rounds of selection we will sequence randomclones and test for binding by ELISA. Following sequence analysis of theimproved clones, all the allowable improved mutations are combined intoa combinatorial beneficial mutagenesis (CBM) library to select forsynergistic improvements to binding of both subtype H5 isolates. The CBMlibrary is made by synthesizing degenerate oligo nucleotides torepresent all improved and original parental residues at all positions.The resulting library is selected under increasing stringencies,similarly to LTM screening. Following sufficient selection the pool issubcloned into a pBAD expression vector to express and purify monomericscFv protein from E. coli for binding and neutralization assays,described above.

H1 neutralizing antibodies can be optimized in an analogous manner. Inthis case one can select and optimize using any reference proteinsequences from 1918, 1976, and current as either a starting point ordestination.

In addition, intertype recognition is tested with the neutralizingantibody clones. An example of intertype recognition is coincidental orengineered H1 binding from an H5 sourced or optimized clone.

The handling of antibody libraries, such as libraries from variousdonors or characterized by reactivity to different isolates of subtypesof a virus, including but not limited to influenza viruses, can begreatly facilitated by applying unique barcodes distinguishing thevarious antibody collections. The barcodes preferably are selected suchthat they are capable of propagating along with the clone(s) labeled.

Thus the barcodes can be non-coding DNA sequences of about 1-24non-coding nucleotides in length that can be deconvoluted by sequencingor specific PCR primers. This way, a collection of nucleic acids, suchas an antibody repertoire, can be linked at the cloning step.

In another example, the barcodes are coding sequences of silentmutations. If the libraries utilize restriction enzymes that recognizeinterrupted palidromes (e.g. Sfi GGCCNNNNNGGCC), distinct nucleotidescan be incorporated in place of the “N's” to distinguish variouscollections of clones, such as antibody libraries. This barcodingapproach has the advantage that the repertoire is linked at theamplification step.

In a different example, the barcodes are coding sequences that encodeimmunologically distinct peptide or protein sequences fused to phageparticles. Examples include, for example, epitope (e.g. Myc, HA, FLAG)fusions to pIII, pVIII, pVII, or pIX phages. The epitopes can be usedsingly or in various combinations, and can be provided in cis (on thelibrary-encoding plasmid) or in trans (specifically modified helperphage) configuration.

Other examples of possible barcodes include, without limitation,chemical and enzymatic phage modifications (for phage libraries) withhaptens or fluorescent chromophores. Such tags are preferred for asingle round of selection.

While barcoding is illustrated herein for distinguishing antibodylibraries, one of ordinary skill will appreciate that the describedapproaches are broadly applicable for uniquely labeling anddistinguishing nucleic acid molecules and collections of nucleic acidsin general.

Epitope Mapping of Neutralizing Antibodies

Once neutralizing antibodies with the desired properties have beenidentified, it might be desirable to identify the dominant epitope orepitopes recognized by the majority of such antibodies. Methods forepitope mapping are well known in the art and are disclosed, forexample, in Morris, Glenn E., Epitope Mapping Protocols, Totowa, N. J.ed., Humana Press, 1996; and Epitope Mapping: A Practical Approach,Westwood and Hay, eds., Oxford University Press, 2001.

Epitope mapping concerns the identification of the epitope to which anantibody binds. There are many methods known to those of skill in theart for determining the location of epitopes on proteins, includingcrystallography analysis of the antibody-antigen complex, competitionassays, gene fragment expression assays, and synthetic peptide-basedassays (see for example, in Chapter 11 of Harlow and Lane, UsingAntibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1999; U.S. Pat. No. 7,332,579, each of whichis incorporated herein by reference in its entirety). An antibody binds“essentially the same epitope” as a reference antibody, when the twoantibodies recognize epitopes that are identical or stericallyoverlapping epitopes. A commonly used method for determining whether twoantibodies bind to identical or sterically overlapping epitopes is thecompetition assay, which can be configured in all number of differentformats, using either labeled antigen or labeled antibody. Usually, anantigen is immobilized on a 96-well plate, and the ability of unlabeledantibodies to block the binding of labeled antibodies is measured usingradioactive or enzyme labels.

Production of Neutralizing Antibodies

Once antibodies with the desired neutralizing properties are identified,such antibodies, including antibody fragments can be produced by methodswell known in the art, including, for example, hybridoma techniques orrecombinant DNA technology.

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster, is immunized to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among thesecell lines, preferred myeloma cell lines are murine myeloma lines, suchas those derived from MOPC-21 and MPC-11 mouse tumors available from theSalk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2or X63-Ag8-653 cells available from the American Type CultureCollection, Rockville, Md. USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); andBrodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay, (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

Recombinant monoclonal antibodies can, for example, be produced byisolating the DNA encoding the required antibody chains andco-transfecting a recombinant host cell with the coding sequences forco-expression, using well known recombinant expression vectors.Recombinant host cells can be prokaryotic and eukaryotic cells, such asthose described above.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)).It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, according to a preferred method, humanized antibodiesare prepared by a process of analysis of the parental sequences andvarious conceptual humanized products using three-dimensional models ofthe parental and humanized sequences.

In addition, human antibodies can be generated following methods knownin the art. For example, transgenic animals (e.g., mice) can be madethat are capable, upon immunization, of producing a full repertoire ofhuman antibodies in the absence of endogenous immunoglobulin production.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:2551 (1993);Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al., Yearin Immuno. 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369 and5,545,807.

Neutralizing Antibodies

A number of neutralizing antibodies have been identified through the useof the techniques described herein, including those described in theExamples below. In one aspect, the present invention providesneutralizing antibodies that bind to a hemagglutinin protein epitope. Inone embodiment, the neutralizing antibody binds to at least one epitopeon the HA1 subunit of the hemagglutinin protein. In another embodiment,the neutralizing antibody binds to at least two, at least three, atleast four, at least five, or at least six epitopes on the HA1 subunitof the hemagglutinin protein. In a preferred embodiment, theneutralizing antibody of the present invention binds to an epitope thatis substantially the same as the epitope for (i) an antibody comprisinga heavy chain amino acid sequence shown as SEQ ID NO: 4 and a lightchain amino acid sequence shown as SEQ ID NO:71 (antibody 1 in theExample below and as shown in Table 1); (ii) an antibody comprising aheavy chain amino acid sequence shown as SEQ ID NO:45 and a light chainamino acid sequence shown as SEQ ID NO:140 (antibody 2 in the Examplebelow and as shown in Table 1); (iii) an antibody comprising a heavychain amino acid sequence shown as SEQ ID NO:9 and a light chain aminoacid sequence shown as SEQ ID NO:81 (antibody 3 in the Examples belowand as shown in Table 1); (iv) an antibody comprising a heavy chainamino acid sequence shown as SEQ ID NO:61 and a light chain amino acidsequence shown as SEQ ID NO:158 (antibody 4 in the Example below and asshown in Table 1); or (v) an antibody comprising a heavy chain aminoacid sequence shown as SEQ ID NO:61 and a light chain amino acidsequence shown as SEQ ID NO:159 (antibody 5 in Table 1); (vi) anantibody comprising a heavy chain amino acid sequence shown as SEQ IDNO:61 and a light chain amino acid sequence shown as SEQ ID NO:160(antibody 6 in Table 1). This is summarized in Table 1 below.

In some embodiments, the antibodies of the present invention neutralizeviruses containing H5 and/or H1. In other embodiments, the antibodiesneutralize both H5 and H1. In one embodiment, the antibodies of thepresent invention do not prevent hemagglutination. In other embodiments,the antibodies do not prevent the binding of an influenza A virus to atarget cell to be infected. In another embodiment, theanti-hemagglutinin antibody does not prevent the receptor binding siteon the globular head region of the HA of an influenza A virus fromattaching to a target cell to allow hemagglutinin activity of HA tooccur.

TABLE 1 Neutralizing Abs Antibody Heavy chain SEQ ID NO: Light chain SEQID NO: Neutralizes 1 QVQLVQSGAEVKKPGSSVRVSCKTSGGTFSSYAVTWVETTLTQSPGTLSLSPGERATLSCRVSQSVSSNYLAWYQQKP H5, H1RQAPGQGLEWMGGIIGMFGTTNYAQKFQGRLTITADEGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEMTSTAYMELSSLRSEDTAVYYCARGSYYYETTLDYW DFAVYYCQQYGTSPRAFGHGTKVEIKRTV GRGTL(SEQ ID NO: 71) (SEQ ID NO: 4) 2 QVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAVTWVETTLTQSPATLSVSPGERATLSCRASQSVSRNLAWYQQKPG H5, H1RQAPGQGLEWMGGIIGMFGTRNYAQKFQGRVTITADEQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDLTSTAYMELSSLRSEDTAVYYCARGSYYYESSLDYWG FAVYYCQQYGSSSITFGQGTRLEIKRTV RGTL(SEQ ID NO: 140) (SEQ ID NO: 45) 3 EVQLVQSGAEVKKPGSSVKVSCKTSGGTFSSYAVTWVQSVLTQPPSVSGAPGQRVTISCGGSRSNIGAGYDVHWYQQ H5, H1RQAPGQGLEWMGAIIGMFGTTNYAQKEQGRVTITADEFPGTAPKVVIYGNNNRPSGVPDRFSGSKSGTSASLAITGLLTSTAYMELSSLRSDDTAVYYCARGSYYYESSLDYWG QAEDEANYYCQSYDTNLGGSIFGGGTQVTVLKGTL (SEQ ID NO: 81) (SEQ ID NO: 9) 4QVQLQESGPGLVKPSETLSLTCTVSGYSFDSGYYWGWNFMLTQPHSVSESPGKTVTISCTGSGGNIARNYVQWYQQR H5LRQPPGKGLEWIGSIYHSRNTYYNPSLKSRVTISVDTPGSAPVTVILEDDKRPSGIPDRFSGSIDRSSNSASLTISGSKNQFSLQLSSVTAADTAVYYCARGTWYSSNLRYWFD LRTEDEALYYCQSYDDSDLVVFGGGTKLTPWGKGTL (SEQ ID NO: 158) (SEQ ID NO: 61) 5QVQLQESGPGLVKPSETLSLTCTVSGYSFDSGYYWGWNFMLTQPHSVSESPGKTVTISCTGSSGSIASNYVQWYQQRP H5LRQPPGKGLEWIGSIYHSRNTYYNPSLKSRVTISVDTGSAPTTVIYEDYQRPSGVPDRFSGSIDSSSNSASLTISGLKSKNQFSLQLSSVTAADTAVYYCARGTWYSSNLRYWFD TEDEADYYCQSYDDSDHLIFGGGTKLTVLPWGKGTL (SEQ ID NO: 159) (SEQ ID NO: 61) 6QVQLQESGPGLVKPSETLSLTCTVSGYSFDSGYYWGWQSVLTQPPSASGTPGQRVTJSCSGSSSNIGSNTVNWYKQLP H5LRQPPGKGLEWIGSIYHSRNTYYNPSLKSRVTISVDTGTAPRLLIYSNDQRPSGVPDRFSGSKSGTSASLAISGLQSESKNQFSLQLSSVTAADTAVYYCARGTWYSSNLRYWFD DEANYYCAAWDDSLSGWVFGGGTKLTVLPWGKGTL (SEQ ID NO: 160) (SEQ ID NO: 61)

Based on the experiments described in the Examples below, a number of H5anti-hemagglutinin antibody heavy chain/light chain pairings wereidentified. As shown in Table 2, column 1 provides the heavy chain aminoacid sequence, column 2 provides the corresponding SEQ ID NO: for theheavy chain sequence, column 3 provides the amino acid sequence forthose light chains that pair with the heavy chains in the same row, andcolumn 4 provides the corresponding SEQ ID NOS: for the light chainsequence. For example, the heavy chain sequence shown as SEQ ID NO:1pairs with the light chain sequence shown as SEQ ID NO:68, SEQ ID NO:2pairs with SEQ ID NO:69, etc. In some embodiments, a heavy chain canpair with more than one light chain. For example, the heavy chainsequence shown as SEQ ID NO:6 pairs with either the light chain sequenceshown as SEQ ID NO:74 or the light chain sequence shown as SEQ ID NO:75;or the heavy chain sequence shown as SEQ ID NO:7 pairs with one of (i)the light chain sequence shown as SEQ ID NO:75, (ii) the light chainsequence shown as SEQ ID NO:76, or (iii) the light chain sequence shownas SEQ ID NO:77.

In one embodiment, the neutralizing antibodies of the present inventioncontain at least one heavy chain polypeptide containing an amino acidsequence shown in Table 2, and/or at least one light chain polypeptidecontaining an amino acid sequence shown in Table 2.

TABLE 2 SEQ SEQ ID ID Heavy Chain Sequence NO Light Chain Sequence NOEVQLVQSGTEVKKPGSSVKLS 1 QSALTQPASVSGSPGQSITISC 68 CKASGGTFSSYAVTWVRQAPGTGTSSDFGGSNHVSWYQQHPGK QGLEWMGGIIGMFGTTNYAQK APKLIIYDVSDRPSGVSNRFSGFQGRLTITADEMTSTAYMELS SKSGNTASLTVSGLQAEDEAHY SLRSEDTAVYYCARGSYYYETYCSSYAGSNNFVFGTGTKVTVL TLDYWGRGTM EVQLVQSGAEVKKPGSSVRVS 2ETTLTQSPGTLSLSPGERATLS 69 CKTSGGTFSSYAVTWVRQAPG CRASQTVSSSYLAWYRQKPGQAQGLEWMGGIIGMFGTTNYAQK PRLLIYGTSSRATGIPDRFSGS FQGRLTITADEMTSTAYMELSGSGTDFTLTISRLEPEDFAVYY SLRSEDTAVYYCARGSYYYET CQQYGNSRVTFGPGTKVDIKRTTLDYWGRGTM V QVQLQQSGAEVKK2GSSVRVS 3 EIVMTQSPGTLSLSPGERATLS 70CKTSGGTFSSYAVTWVRQAPG CRASQSLSGSNVAWYQQKFGQA QGLEWMGGIIGMFGTTNYAQKPRLLIHGASKRAAGIPDRFSGS FQGRLTITADEMTSTAYMELS GSGTDFTLTISRLQPDDYAVYYSLRSEDTAVYYCARGSYYYET CQQYGTKPFTFGQGSKLEIKRT TLDYWGQGTM VQVQLVQSGAEVKKPGSSVRVS 4 ETTLTQSPGTLSLSPGERATLS 71 CKTSGGTFSSYAVTWVRQAPGCRVSQSVSSNYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK PRLLIYGASSRATGIPDRFSGSFQGRLTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY SLRSEDTAVYYCARGSYYYETCQQYGTSPRAFGHGTKVEIKRT TLDYWGRGTL V QVQLQQSGAEVKKPGSSVRVS 5QSALTQPPSASGSPGQSVTISC 72 CKTSGGTFSSYAVTWVRQAPG TGASSDIGGYKSVSWYQQHPGKQGLEWMGGIIGMFGTTNYAQK APKLIIYDVTERPSGVPDRFSA FQGRLTITADEMTSTAYMELSSKSGNTASLTVSGLQAEDEADY SLRSEDTAVYYCARGSYYYET YCSSYGGSNNLVVFGGGTKVTVTLDYWGKGTL L DIQMTQSPSSVSASVGDRVTIT 73 SWLAWYQQKPGKAPKLLIYAASTLQRGVPSRFSGSGSGTDFTLT INSLQPEDFATYYCQQYNSYPL TFGGGTKVEIKREVQLVQSGAEVKKPGSSVRVS 6 EIVLTQSPGTLSLSPGERATLS 74 CKTSGGTFSSYAVTWVRQAPGCRASQSVSNNYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQK PRLLIYGASSRATGIPDRFSGSFQGRLTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYY SLRSEDTAVYYCARGSYYYETCQQYGRSPRTFGQGTKVEIKRT TLDYWGKGTL ETTLTQSPGTLSLSPGERATLS 75CRASQSLGGANLGWYQQKFGQP PRLLIYGASSRATGVPDRESGS GSGTDFALTISRLEPEDFAVYYCQQYGSKPYTFGQGTKLEIKRT V EVQLVQSGAEVKKPGSSVRVS 7 ETTLTQSPGTLSLSPGERATLS75 CKTSGGTFSSYAVTWVRQAPG CRASQSLGGANLGWYQQKFGQP QGLEWMGGIIGMFGTTNYAQKPRLLIYGASSRATGVPDRFSGS FQGRLTITADEMTSTAYMELS GSGTDFALTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYET CQQYGSKPYTFGQGTKLEIKRT TLDYWGQGTL VETTLTQSPATLSVSPGERATLS 76 CRASQSVSTNLAWYQQKPGQAP RLLIHGASTRATGIPARESGSGSGTEFTLTISSLQSEDSAVYYC QQHNNWPPVTFGRGTKVEIKRT V ETTLTQSPATLSVSPGERATLS77 CRASQSVSRNLAWYQQKPGQAP RLLIYGASSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYGSSSITFGQGTRLEIKRTV V EVQLVQSGAEVKKPGSSVKVS 8 QSVLTQPPSASGAPGQRVTISC78 CKTSGGTFSSYAVTWVRQAPG TGSSSNIGAGYDVHWYQQLPGR QGLEWMGAIIGMFGTTNYAQKAPKLLIYGNSNRPSGVPARFSG FQGRVTLTADELTSTAYMELS SKSATSASLAITGLQAEDEADYSLRSEDTAVYYCARGSYYYES YCQSYDSSLSGVVFGGGTKLTV SLDYWGRGTL LEIVMTQSPATLSVSPGERAILS 79 CRASRSVSTNLAWYQQKPGQAP RLLIYGASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQYGSSPNFGGGTKVEIKR EVQLVQSGAEVKKPGSSVKVS 9EIVMTQSPGTLSLSPGERATLS 80 CKTSGGTFSSYAVTWVRQAPG CRASQSVPNRYIAWYQQKPGQAQGLEWMGAIIGMFGTTNYAQK PRLLTYGASSRATGIPDRFSGS FQGRVTITADELTSTAYMELSGSGPDFTLTISRLEPEDFAVYY SLRSDDTAVYYCARGSYYYES CQQYGRSPQTFGQGTKLETKGTSLDYWGKGTL V QSVLTQPPSVSGAPGQRVTISC 81 GGSRSNIGAGYDVHWYQQFPGTAPKVVIYGNNNRPSGVPDRESG SKSGTSASLAITGLQAEDEANY YCQSYDTNLGGSIEGGGTQVTV LEIVMTQSPGTLSVSPGDAATLS 82 CRASRNINNNLAWYQQTPGQAP RLLIYGASTRATGLPARFTGSGSGTEFTLTISSLQSEDFAVYYC QQYNNWPRTPGQGTKVEIKR EVQLVQSGTEVKKPGSSVKVS 10ETTLTQSPGTLSLSPGERATLS 83 CKVSGGTFSSYAVTWVRQAPG CRASQIVDSSYLAWYQHRPGQAQGLEWMGAIIGMFGTTNYAQK PRILIYGASSRAPGVPDRFSGS FQGRVTITADELTSTAYMELSGSGTDFTLTISRLEPEDFAVYY SLRSDDTAVYYCARGSYYYES CQQYAVSPRTFGQGTKVEIKRTSLDYWGGGTT V EVQLVQSGAEVKKPGSSVKVS 11 EIVLTQSPGTLSLSPGDRATLS 84CKTSGGTFSSYAVTWVRQAPG CRASQSLGTNYLAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQKPRLLIYGASSRATGIPDRFSGS FQGRVTITADELTSTAYMELS GSGTDFTLIISRLEPEDFAVYYSLRSDDTAVYYCARGSYYYES CQQYGRSPQKFGQGTKVEIKRT SLDYWGQGTL VQSVLTQPPSASGTPGQRVTISC 85 SGSSSNIGSNYVYWYQQLPGTA PKLLIYRINNQRPSGVPDRFSGSKSGTSASLATSGLRSEDEANY YCAAWDDSLSGWVFGGGTKLTV L EIVMTQSPATLSVSPGERAILS86 CRASRSVSTNLAWYQQKPGQAP RLLTYGASTRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYGSSPNFGGGTKVEIKR EVQLVQSGAEVKKPGSSVKVS 12 DIQMTQSPSSVSASVGDRVTTT 87CKTSGGTESSYAVTWVRQAPG CRASQGISSWLAWYQQKPGKAP QGLEWMGAIIGMFGTTNYAQKKLLIYAASSLQSGVPSRFSGSG FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDVATYYCSLRSDDTAVYYCARGSYYYES QKYNSAPRTFGQGTKVEIKR SLDYWGQGTMQVQLVQSGAEVKKPGSSVKVS 13 DIQLTQSPSSLSASVGDRVTIT 88 CKTSGGTFSSYAVTWVRQAPGCRASQSISNYLNWYQQKPGKAP QGLEWMGAIIGMFGTTNYAQK KLLIYAASSLQSGVPSRESGSGFQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDSATYYC SLRSDDTAVYYCARGSYYYESQQSHSTPRTFGQGTKLEIKRTV SLDYWGQGTM QAVLTQPPSASGTPGQRVTISC 89SGSSSNIGTNTVNWYQQLPGTA PKLLIYRNIQRPSGVPDRFSGS KSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVL EVQLVQSGAEVKKPGSSVKVS 14 EIVLTQSPGTLSLSPGEKATLS90 CKTTGGTFSSYAVTWVRQAPG CRASQSVSNTYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQKPRLLLYGASSRAPGIPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISRLEAEDFAVYFSLRSEDTAVYYCARGSYYYES CQQYAGSPRTFGQGTKVEIKRT SLDYWGQGTL VEIVLTQSPGSLSLSPGERATLS 91 CRASQSVSHGYLAWYQQKPGQA PRFLIYGASSRPTGIPDRFRGSGSGTDFTLTISSLEPEDSAVYY CQQYSTSPLTFGGGTKVETKRT V PELTQPPSASGTPGQRVTISCS92 GSSSNIGSNYVYWYQQLPGTAP KLLIYRNNQRPSGVPDRFSGSK SGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL EVQLVQSGAEVKKPGSSVKVS 15 ETTLTQSPGTLSLSPGERATLS 93CKTTGGTFSSYAVTWVRQAPG CRASQIVDSSYLAWYQHRPGQA QGLEWMGGIIGMFGTTNYAQKPRILIYGASSRAPGVPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYGSPPRTFGQGTKVEIKRT SLDYWGQGTM VEVQLVQSGAEVKKPGSSVKVS 16 QSVLTQPPSTSGTPGQRVTISC 94 CKTSGGTFSSYAVTWVRQAPGSGSSSNIGRKTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQK PKLLIYNDNQRPSGVPDRFSGSFQGRVTLTADELTSTAYMELS KAGTSASLAISGLQSEDEADYY SLRSEDTAVYYCARGSYYYESCAVWDDSLNAWVFGGGTKLTVL SLDYWGKGTL EVQLVQSGAEVKKPGSSVKVS 17DVVMTQSPLSLPVTPGEPAAIS 95 CKTSGGTFSSYAVTWVRQAPG CRSSQSLLHSDGNNYLDWYLQKQGLEWMGAIIGMFGTTNYAQK PGQSPHLLIYLGSNRASGVPDR FQGRVTLTADELTSTAYMELSFSGSGSGTDFELKISRVEAEDA SLRSEDTAVYYCARGSYYYES GVYYCMQASQTPRTFGQGTKLESLDYWGKGTM LKRTV EVQLVQSGAEVKKPGASVKVS 18 HVILTQPPSVSVAPGMTARMTC 96CKASGGAFSSYAVTWVRQAPG GGDNVGRRNVHWYQQKPGQAPV QGLEWMGGIIGMFGTTNYAQKLVVYDDGGRPSAIPARFSGSKS FQGRVTITADELTSTAYMELS GNTATLIISRVEAGDEADYYCQSLRSEDTAVYYCARGSYYYES MWHSSGDQWVFGGGTKLTVL SLDYWGQGTMEVQLVQSGAEVKKPGSSVKVS 19 ETTLTQSPGTLSLSPGERATLS 97 CKTSGGTFSSYAVTWVRQAPGCRASQSISPNYLAWYQQRPGQA QGLEWMGGIIGMFGTTNYAQK PRLLIYGASKRATGIPDRFSGSFQGRVTMTADEMTSTAYMELS GSGTDFTLTISSLEPEDSAVYY SLRSEDTAVYYCARGSYYYESCQHQGFGQGTKVEIKRTV SLDYWGKGTL EVQLVQSGAEVKKPGSSVKVS 20QSVLTQPPSASGTPDQRVTISC 98 CKTSGGTFSSYAVTWVRQAPG SGSGSNIGSNYVYWYQQLPGAAQGLEWMGGIIGMFGTTNYAQK PKLLMSRNNQRPSGVPDRFSGS FQGRVTITADEMTSTAYMELSKSGTSASLAISGLQSEDEADYY SLRSEDTAVYYCARGSYYYES CAAWDDSLTGYVFGTGTKLTVLSLDYWGRGTL EVQLVQSGAEVKKPGSSVKVS 21 SYVLTQPPSVSVAPGKTARITC 99CKTSGGTFSSYAVTWVRQAPG GGKNIGSKSVHWYQQKSGQAPV QGLEWMGGIIGMFGTTNYAQKLVIYGDSDRPSGIPERFSGSNS FQGRVTITADEMTSTAYMELS GNTATLTISGVEAEDEADYYCQSLRSEDTAVYYCARGSYYYES VWDNTSDHAGVFGGGTKVTVL SLDYWGRGTMEVQLVQSGAEVKKPGSSVKVS 22 QSVLTQPPSASGTPGQRVTISC 100CKTSGGTFSSYAVTWVRQAPG SGSSSNIGSNYVYWYQQLPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIYRNNQRSSGVPDRFSGS FQGRVTLTADELTSTAYMELS KSGTSASLAISGLRSEDEADYYSLRSEDTAVYYCARGSYYYES CAAWDDSLSGLVFGGGTKLTVL SLDYWGQGTLQSVLTQPPSVSAAPRQSVTISC 101 SGTTSNIGNNPVSWYQQFPGRA PNLLIYYNDVVPSGVSDRFSASKAGTSASLAISRLQSEDEADYY CATWDDSLSAWVFGGGTQLTVL QAVLTQPPSASGTPGQRVTISC 102SGSSSNIGSNYVYWYQQLPGTA PKLLIYSNNQRPSGVPDRFSGS KSGTSASLVISGLQSEDETDYYCAAWDDSLNGWVFGGGTKLTVL EVQLVQSGAEVQKPGSSVKVS 23 QSALTQPPSASGSPGQSVTITC103 CKTSGGTFSSYAVTWVRQAPG AGASSDLGDYKSVSWYQQHPGK QGLEWMGAIIGMFGTTNYAQKAPKLIIYDVIKRPAGVPDRFSA FQGRVTLTADELTSTAYMELS SKSGNTASLTVSGLQAEDEADYSLRSEDTAVYYCARGSYYYES YCSSYAGSNNIVIEGGGTKLTV SLDYWGQGTL LEVQLVQSGAEVKKPGSSVKVS 24 ETTLTQSPGTLSLSPGERATLS 104CKTSGGTFSSYAVTWVRQAPG CRASQGIDRKYLAWYQRKHGQA QGLEWMGGIIGMFGTTNYAQKPRLLIYGASNRATGIPDRFSGS FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYADSFVSFGQGTKLEIKRT SLDYWGQGTL VEVQLVQSGAEVKKPGSSVKVS 25 DIQLTQSPSTLSASVGDRVTIT 105CKASGGTFSSYAISWVRQAPG CRASQSISRWLAWYQQKPGKTP QGLEWMGAIIGMFGTTNYAQKKLLIYEASNLQSGVPSRFSGSG FQGRVTITADELTSTAYMELS SGTEFTLTISSLQPDDFATYYCSLRSDDTAVYYCARGSYYYES QQYKSDFLVTFGPGTKVDIKRT SLDYWGQGTL VEVQLVQSGAEVKKPGSSVKVS 26 QSVLTQPPSVSGAPGQKITISC 106CKTSGGTFSSYAVTWVRQAPG TGSSSNIGTGYDVHWYQQLPGR QGLEWMGAIIGMFGTTNYAQKAPRLLISADANRPSGVPDRFSA FQGRVTLTADQLTSTAYMELS SKSGTSASLAITGLQAEDEADYSLRSEDTAVYYCARGSYYYES YCQSYDTRLGGSIFGGGTQLTV SLDYWGRGTL LEVQLVQSGAEVKKPGSSVKVS 27 EIVMTQSPGTLSVSPGDAATLS 107CKTSGGTFSSYAVTWVRQAPG CRASRNINNNLAWYQQTPGQAP QGLEWMGAIIGMFGTTNYAQKRLLIYGASTRATGLPARFTGSG FQGRVTITADELTSTAYMELS SGTEFTLTISSLQSEDFAVYYCSLRSDDTAVYYCARGSYYYES QQYNNWPRTFGQGTKVEIKR SLDYWGKGTLEVQLVQSGAEVKKPGSSVKVS 28 QSVLTQPPSVSAAPGQEVTTTC 108CKTSGGTFSSYAVTWVRQAPG SGSGANIGNNYVSWYQQVPGTA QGLEWMGAIIGMFGTTNYAQKPKLVIYDNNRRPSGIPDRFSGS FQGRVTITADELTSTAYMELS KSGTSATLGITGLQTGDEADYYSLRSDDTAVYYCARGSYYYES CGTWDSSLSAVVFGGGTKVTVL SLDYWGKGTTEVQLVQSGAEVKKPGSSVKVS 29 QAVLTQPPSASGTPGQTVTISC 109AVTWVRQAPGQGLEWMGGIIG SGVTSNIGNNYVYWYQQLPGTA MFGTTNYAQKFQGRVTITADEPRLLIYSNNQRPSGVPDRFSGS LTSTAYMELSSLRSEDTAVYY KSGTSASLAISGLRSEDEADYYCARGSYYYESSLDYWGKGTL CSAWDDSLRENLFGTGTQLTVL EVQLVQSGAEVKKPGSSVKVS 30QAVLTQPPSASGTPGQRVTISC 110 CKTSGGTFSSYAVTWVRQAPG SGSSSNFGMNAVNWYQQLPGTAQGLEWMGAIIGMFGTTNYAQK PKLLMYSNSKRPSGVPDRFSGS FQGRVTLTADELTSTAYMELSKSGTSASLAISGLQSEDEADYY SLRSEDTAVYYCARGSYYYES CSAWDDNLNGWVEGGGTKVTVLSLDYWGRGTM EVQLVQSGAEVKKPGSSVKVS 31 QAVLTQPPSASGTPGQRVTISC 111CKTSGGTFSSYAVTWVRQAPG SGSSSNIGSNTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIYRNNQRPSGVPDRFSGS FQGRVTITADELTSTAYMELS KSGTSASLAISGLQSEDEADYYSLRSDDTAVYYCARGSYYYES CATWDDSLTSVVFGGGTKVTVL SLDYWGKGTMVTVSQMQLVQSGAEVKKPGSSVKVS 32 ETTLTQSPSSLSASIGDRITIA 112CKTSGGTFSSYAVTWVRQAPG CQASQDIRNRLNWYLQRPGKAP QGLEWMGAIIGMFGTTNYAQKQLLIYDASNLETGVPSKFAGRG FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDIGTYFCSLRSDDTAVYYCARGSYYYES QQYGDLSPLTFGGGTKVDIRRT SLDYWGKGTM VQMQLVQSGAEVKKPGSSVKVS 33 ETTLTQSPSSLSASIGDRITIA 113CKTSGGTFSSYAVTWVRQAPG CQASQDIRNRLNWYLQRPGKAP QGLEWMGAITGMFGTTNYAQKQLLIYDASNLETGVPSKFAGRG FQGRVTLTADELTSTAYMELS SGTDFTLTISSLQPEDIGTYFCSLRSEDTAVYYCARGSYYYES QQYGDLSPLTFGGGTKVDIRRT SLDYWGQGTLVTVS VDIQMTQSPSSLSASVGDRVTIT 114 CRASQSISNYLNWYQQKPGKAP KLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDSATYYC QQSHSTPRTFGQGTKLEIKRTV QVQLVQSGAEVKKPGSSVKVS 34QSVLTQPPSVSGAPGQRVTISC 115 CKTTGGTFSSYAVTWVRQAPG TGTSSNIGAGFDVHWYQQFPGTQGLEWMGGIIGMEGTTNYAQK APKLLIYDNVKRPSGVPDRFSG FQGRVTITADEMTSTAYMELSSKSGTSASLAITGLRAEDEADY SLRSEDTAVYYCARGSYYYES YCQSYDTSLSRYVEGTGTKVTVSLDYWGKGTM L QVQLVQSGAEVKKPGSSVKVS 35 ETTLTQSPGTLSLSPGERATLS 116CKASGGTFSSYAVTWVRQAPG CRASQSVSSIYLAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQKPRLVIHGASSRATGIPDRFSGS FQGRVTLTADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYGSSPLTFGGGTKVEIKRT SLDYWGQGTL VQVQLVQSGAEVKKPGSSVKVS 36 ETTLTQSPGILSLSPGESATLS 117CKTSGGTFSSYAVTWVRQAPG CGASQTISSRYLAWYQQRPGQA QGLEWMGAIIGMFGTTFNYAQPRLLIFDASRRATGVPDRFSGG KFQGRVTLTADELTSTAYMEL GSGTDFTLTISRLEPEDFGVFYSSLRSEDTAVYYCARGSYYYE CQQYGISPYTFGQGTKLEIKRT SSLDYWGQGTL VEIVLTQSPGTLSLSPGERATLS 118 CRASQSVSNNLAWYQQKFGQAP RLLIYAASSRATDIPARFSGSGSGTDFTLTISRLEPEDFAVYYC QQYVDSPRTFGQGTKVEIKRTV EIVLTQSPGTLSLSPGERATLS 119CRASQSVSSSYLAWYQQKPGQA PRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT V EIVLTQSPGTLSLSPGERATLS 120CRASQSVSSSYLAWYQQKPGQA PRLLIYGASSRAAGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQYGSSPKTFGQGTKVEIKRT V QSVLTQPPSVSGAPGQGVTISC 121SGSSSNIGANYVVHWYRQLPGA APKLLIYDDIHRPSGVPDRFSG SRSGTSASLAITGLQPEDEADYYCQTYDTSLRGSVFGGGTKLTV L QSVLTQPPSASGTPGQRVTISC 122SGSSSNIGSNYVYWYQQLPGTA PKLLIYRNNQRPSGVPDRFSGS KSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKVTVL QAVLTQPPSVSVAPGKTATITC 123 GVNNLGRKSVHWYQQKPGQAPVLVVYDSNDRPSGIPERFSGSNS GNTATLIISRVEAGDEADYSCQ VWDNNVDHPVFGGGTKLTVLHVILTQPPSVSVAPGMTARMTC 97 GGDNVGRRNVHWYQQKPGQAPV LVVYDDGGRPSAIPARFSGSKSGNTATLIISRVEAGDEADYYCQ MWHSSGDQWVFGGGTKLTVL SSELTQDPAVSVALGQTVRITC 124QGDSLRSYYASWYQQKPGQAPV LVIYGKNNRPSGIPDRFSGSSS GNTASLTITGAQAEDEADYYCNSRDSSGNHWVFGGGTKVTVL EIVMTQSPGTLSLSPGERATLS 125 CRASQSVSSSLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSG SGTDFTLTISRLEPEDEAVYYC QQYGGSPRTFGQGTKLEIKREIVLTQSPGTLSLSPGERATLS 126 CRASQSVSSSYLAWYQQKPGQA PRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPNFGGGTKVDIKR QAVLTQPPSASGTPGQRVTISC 127SGSSSNIGSNSVYWYQQLPGTA PKLLIYRINNQRPSGVPDRFSG SKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTV L QSVVTQPPSVSGAPGQRVTISC 128TGSSSNIGAGYDVHWYQQLPGT APKLLIYGDTNRPSGVPDRFSG SKSGTSASLAITGLQAEDEADYYCQSYDGSRSGLVFGGGTKLTV L QVQLVQSGAEVKKPGSSVKVS 37 EIVLTQSPGTLSLSPGERATLS129 CKTTGGTFSSYAVTWVRQAPG CRASQSLSNAYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQKPRLLLYGGSTRATGIPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISSLEAEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYGSSPRTFGQGTKVEIKRT SLDYWGKGTL VQVQLLQSGAEVKKPGSSVKVS 38 EIVLTQSPGTLSLSPGERATLS 130CKTTGGTFSSYAVTWVRQAPG CRASQSVSSSYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQKPRLLIYGTSSRATDIPDRFSGS FQGRVTITADEMTSTAYMELS GSGTDFTLTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYGRSPFTFGGGTKVEIKRT SLDYWGKGTL VQVQLQQSGAEVKKPGSSVKVS 39 DIQLTQSPSSLSASVGDRVTIT 131CKTSGGTFSSYAVTWVRQAPG CRASQGISNYLAWYQQKPGKVP QGLEWMGAIIGMFGTTNYAQKKLLIYAASTLQSGVPSRFSGSG FQGRVTLTADELTSTAYMELS SGTDFTLTISSLQPEDVATYYCSLRSEDTAVYYCARGSYYYES QKYNSAPRTFGQGTKVEIKRTV SLDYWGQGTLQVQLVQSGAEVKKPGSSVKVS 40 DIQLTQSPSSLSASVGDRVTIT 132CKTTGGTFSSYAVTWVRQAPG CRASQGISNYLAWYQQKPGKVP QGLEWMGGIIGMFGTTNYAQKKLLIYAASTLQSGVPSRFSGSG FQGRVTITADEMTSTAYMELS SGTDFTLTISSLQPEDVATYYCSLRSEDTAVYYCARGSYYYES QKYNSAPLTFGGGTKVEIKRTV SLDYWGQGTLEIVMTQSPGTLSLSPGERATLS 133 CRASQSVSSSYLAWYQQKPGQA PRLLTYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYY CQQYGSSPYTFGQGTKVEIK QVQLVQSGAEVKKPGSSVKVS 41QSVLTQPPSASGTPGQRVTISC 134 CKTSGGTFSSYAVTWVRQAPG SGSSSNIGSNFVYWYQQLPGTAQGLEWMGAIIGMFGTTNYAQK PKLLIYKSNQRPSGVPDRFSGS ) FQGRVTLTADELTSTAYMELSKSGTSASLAISGLRSEDEADYY SLRSEDTAVYYCARGSYYYES CAAWDDSLSGYVFGTGTQLTVLSLDYWGQGTM QVQLVQSGAEVKKPGSSVKVS 42 ETTLTQSPDTLSLSPGERATLS 135CKTSGGSFSSYAVTWVRQAPG CRASQSVSSGSLAWYQQKPGQA QGLEWMGGTIGMFGTTNYAQKPRLLIYAASSRAAGIPDRESGS FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYGSSPGLTFGGGTQVEIKR SLDYWGQGTM TVQVQLVQSGAEVKKPGSSVKVS 43 ETTLTQSPGTLSLSPGERATLS 136CKTSGGTFSSYAVTWVRQAPG CRASQSVSSSYLAWYQQKPGQA QGLEWMGGIIGMFGTTNYAQKPRLLIYGASSRATGIPDRFSGS FQGRVTITADELRSTAYMELS GSGTDFTLTISRLEPEDFAVYYSLRSEDTAVYYCARGSYYYES CQQYGSSPYTEGQGTKLEIKRT SLDYWGQGTL VQVQLVQSGAEVKKPGSSVKVS 44 ETVLTQSPGTLSLSPGERATLS 137CKTSGGTFSSYAVTWVRQAPG CRASQNIGWYLAWYQHKPGQAP QGLEWMGGIIGMFGTTNYAQKRLIMYDASTRATGIPDRFSGSG FQGRVTITADELTSTAYMELS SGTDFTLTISRLEPEDFAVYYCSLRSEDTAVYYCARGSYYYES QQYDSVPSTEGQGTNLEIKRTV SLDYWGQGTLQSVLTQPPSVSGAPGQRVTISC 138 TGSSSNIGAGYDVHWYQQLPGT TPKLLIYDNTNRPSGVPDRFSASKSGASASLAITGLRDEDEADY YCQSYDSSLSASVFGGGTKLTV L QVQLVQSGAEVKKPGSSVKVS 45QSALTQPRSVSGSPGQSVTISC 139 CKTSGGTFSSYAVTWVRQAPG TGTTSDVGGYNYVSWYQQHPGEQGLEWMGGIIGMFGTTNYAQK APKLIIYDVSNRPSGVSNRFSG FQGRVTITADELTSTAYMELSSKSGNTASLTVSGLQAEDEADY SLRSEDTAVYYCARGSYYYES YCSSEAGSSNLIFGGGTKLTVLSLDYWGRGTL ETTLTQSPATLSVSPGERATLS 140 CRASQSVSRNLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYC QQYGSSSITFGQGTRLEIKRTVQVQLVQSGAEVKRPGSSVKVS 46 SYELTQPPSASGTPGQRVTISC 141CKASGGTFSSYAVTWVRQAPG SGSSSNIGSNTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIYSNNHRPSGVPDRFSGS FQGRVTLTADELTSTAYMELS KSGTSASLAISGLQSEDEADYYSLRSEDTAVYYCARGSYYYES CATWDDSLNGWVFGGGTKVTVL SLDYWGQGTMQVQLVQSGAEVKKPGSSVKVS 47 QSVLTQPPSASGTPGQRVTISC 142CKASGGTFSSYAVTWVRQAPG SGSSSNIGGNTVNWYQQVPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIHSNNQRPSGVPDRESGS FQGRVTITADELTSTAYMELS KSGTSASLAISGLLSEDEADYYSLRSDDTAVYYCARGSYYYES CEVWDDSLNGRVEGGGTKLTVL SLDYWGQGTLQVQLVQSGAEVKKPGSSVKVS 48 DIQLTQSPSSLSASVGDRVTIT 143CKTSGGTFSSYAVTWVRQAPG CRASQGISNYLAWYQQKPGKVP QGLEWMGAIIGMFGTTNYAQKKLLIYAASTLQSGVPSRFSGSG FQGRVTITADELTSTAYMELS SGTDFTLTISSLQPEDFATYYCSLRSDDTAVYYCARGSYYYES QQSYSTPRTFGQGTKLETKR SLDYWGQGTLHPELTQPPSASGTPGQRVTISC 144 SGSSSNIGSNTVNWYQQLPGTG PKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYY CAAWDDSLNGWVFGGGTKVTVL EIVLTQSPGTLSLSPGERATLS 145CRASQSVSSSYLAWYQQKPGQA PRLLIYGASSRATGIPDRFSGS GSGTDFTLTISRLEPEDFAVYYCQQFGSSQVTFGGGTKVEIKR GVQLVQSGAEVKKPGSSVKVS 49 HVILTQPPSVSVAPGMTARMTC146 CKTSGGTFSSYAVTWVRQAPG GGDNVGRRNHWYQQKPGQAPVL QGLEWMGAIIGMFGTTNYAQKVVYDDGGRPSAIPARFSGSKSG FQGRVTLTADELTSTAYMELS NTATLIISRVEAGDEADYYCQMSLRSEDTAVYYCARGSYYYES WHSSGDQWVFGGGTKLTVL SLDYWGKGTMGVQLVQSGAEVKKPGSSVKVS 50 QAVLTQPPSASGTPGQRVTISC 147CKTSGGTFSSYAVTWVRQAPG SGSSSNIGSNYVYWYQHLPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIDRNDQRPSGVPDRFSGS FQGRVTITADELTSTAYMELS KSGTSASLAISGLRSEDEADYYSLRSDDTAVYYCARGSYYYES CAAWDDNLSGVVFGGGTKVTVL SLDYWGRGTLQVQLVQSGAEVKKPGSSVKVS 51 QSVVTQPPSVSGAPGQRVTISC 148CKASGGTFSSYAISWVRQAPG TGSSIGAGYDVHWYQQLPRTAP QGLEWMGAIIGMFGTTNYAQKKLLIFGNTNRPSGVPDRFSGSK FQGRVTLTADELTSTAYMELS SGTSASLTITGLQAEDEANYYCSLRSEDTAVYYCARGSYYYES QTYDSSLSGWVFGGGTKLTVL SLDYWGKGTMQVQLVQSGAEVKKPGSSVKVS 52 PELTQPPSASGTPGQRVTISCS 149CKASGGTFSSYAISWVRQAPG GSSSNIGSNHVYWYQQLPGTAP QGLEWMGAIIGMFGTTNYAQKKLLIYRNNQRPSGVPDRFSGSK FQGRVTITADELTSTAYMELS SGTSASLAISGLRSEDEADYYCSLRSDDTAVYYCARGSYYYES ATWDDNLSGRLVEGGGTKLTVL SLDYWGKGTMQVQLVQSGAEVKKPGSSVKVS 53 QSVLTQPPSVSGAPGQRVTISC 150CKTTGGTFSSYAVTWVRQAPG IGSNSNIGANFAVHWYQQLPGA QGLEWMGGIIGMFGTTNYAQKAPKLLIYDNTNRPSGVPDRFSG FQGRVTITADEMTSTAYMELS SKSGTSASLDITGLQADDEADYSLRSEDTAVYYCARGSYYYES YCQSYDARLNGWVFGGGTKLTV SLDYWGQGTM LQVQLQQSGAEVKKPGSSVKVS 54 QSALTQPPSASGSPGQSVTISC 151CKTSGGTFSSYAVTWVRQAPG AGASSDIGTYNSVSWYQQHPGK QGLEWMGAIIGMFGTTNYAQKAPKLIIYEVTKRPSGVPDRFSG FQGRVTITADELTSTAYMELS SKSGNTASLTVSGLQAEDEADYSLRSDDTAVYYCARGSYYYES YCNSYAGTKGYVFGSGTKVTVL SLDYWGKGTMQVQLQQSGAEVKKPGSSVKVS 55 SSELTQDPAVSVALGQTVRITC 152CKTSGGTFSSYAVTWVRQAPG QGDSLRNEYASWYQQKPGQAPV QGLEWMGGITGMFGTTNYAQKLVMKGNNNRPSVIPDRFSGSRS FQGRVTITADELTSTAYMELS GNTASLTITGAQAEDEADYYCSSLRSEDTAVYYCARGSYYYEN SRDSSGNRFFGSGTKVTVL SLDYWGKGTLQVQLVQSGAEVKKPGSSVKVS 56 HPELTQPPSLSVSPGQTATISC 153CKTSGGTFSSYAVTWVRQAPG SGERLTNKYTSWYQQRPGQSPA QGLEWMGAIIGMFGTTNYAQKLVIYQDDKRPSGIPERFSGSNS FQGRVTLTADELTSTAYMELS GNTATLTISGTQPMDEAVYYCQSLRSEDTAVYYCARGSYYYES AWDTNTQMTFGGGTKLTVL SLDYWGKGTMQVQLVQSGAEVKKPGSSVKVS 57 QAVLTQPPSASGTPGQRVTISC 154CKTSGGTFSSYAVTWVRQAPG SGSSSNIGTNTVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIYRNIQRPSGVPDRFSGS FQGRVTITADELTSTAYMELS KSGTSASLAISGLQSEDEADYYSLRSDDTAVYYCARGSYYYES CAAWDDSLNGYVFGTGTKLTVL SLDYWGQGTMQVQLVQSGAEVKKPGSSVKVS 58 ETTLTQSPGTVSLSPGERATLS 155CKTSGGTFSSYAVTWVRQAPG CRASQSVGGSNLAWYQQKPGQA QGLEWMGAIIGMFGTTNYAQKPRLVIYATSRKANGIPDRFSGS FQGRVTITADELTSTAYMELS GSGTDFTLTISRLEPEDFAVYFSLRSDDTAVYYCARGSYYYES CQQYGTSPPSVTFGGGTKVEIR SLDYWGRGTM RQVQLVQSGAEVKKPGSSVKVS 59 QAVLTQPPSASGTPGQRVTISC 156CKASGGTFSSYAVTWVRQAPG SGSSSNIGSNPVNWYQQLPGTA QGLEWMGAIIGMFGTTNYAQKPKLLIYSNNQRPSGVPDRFSGS FQGRVTLTADELTSTAYMELS KSGTSASLAISGLQSEDEADYYSLRSEDTAVYYCARGSYYYES CAAWDDSLTGYVFGTGTQLTVL SLDYWGKGTMQVQLVQSGAEVKKPGSSVKVS 60 SYVLTQPPSASGTPDQRVTISC 157CKASGGTFSSYAISWVRQAPG SGSSSNIGSNYVYWYQQFPGAA QGLEWMGGIIGMFGTTNYAQKPKLLMSRNNQRPSGVPDRFSGS FQGRVTITADELTSTAYMELS KSGTSASLAISGLRSEDEAYYSSLRSEDTAVYYCARGSYYYES CAAWDDSLNGLVFGGGTKVTVL SLDYWGKGTTQVQLQESGPGLVKPSETLSLT 61 NFMLTQPHSVSESPGKTVTISC 158CTVSGYSFDSGYYWGWLRQPP TGSGGNIARNYVQWYQQRPGSA GKGLEWIGSIYHSRNTYYNPSPVTVILEDDKRPSGIPDRPSGS LKSRVTISVDTSKNQFSLQLS IDRSSNSASLTISGLRTEDEALSVTAADTAVYYCARGTWYSSN YYCQSYDDSDLVVFGGGTKLT LRYWFDPWGKGTLNFMLTQPHSVSESPGKTVTISC 159 TGSSGSIASNYVQWYQQRPGSA PTTVIYEDYQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEAD YYCQSYDDSDHLIFGGGTKLTV L QSVLTQPPSASGTPGQRVTISC160 SGSSSNIGSNTVNWYKQLPGTA PRLLIYSNDQRPSGVPDRPSGS KSGTSASLAISGLQSEDEANYYCAAWDDSLSGWVFGGGTKLTVL PELTQPHSVSESPGKTVTISCT 161 GSGGRIATNHVQWYQQRPGSAPTIVIYENNQRPSGVPNRFSGSI DDSSNSASLTISALRTEDEADY YCQSADATNVFFGGGTKVTVLPELTQPPSASGTPGQRVTISCS GSSSNIGSNTVNWYQQLPGTAP KLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGWVFGGGTKLTVL DIQMTQSPSSLSAFVGDRVTIT 163CQASQDISNYLNWYQQKPGKAP KLLIYDATNLETGVPSRFSGSG SGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVDIKR QVQLQESGPGQVKYSETLSLT 62 QSVLTQPPSASGTPGQRVTLSC 164CTVSGYSFDSGYYWGWLRQPP SGSSSNIGGNSVNWYQHVPGTA GKGLEWIGSIYHSRNTYYNPSPKLLMHSDDQRPSGVPDRFSGS LKSRVTISVDTSKNQFSLQLS KSGTSASLAISGLQSEDEADYYSVTAADTAVYYCARGTWYSSN CAAWDDSLNAWVFGGGTKVTVL LRYWFDPWGKGTTEVQLVQSGAAVKKPGSSVKVS 63 ETTLTQSPGTLSLSPGERATLS 165CKASGGRFSSYAINWVRQAPG CRASQSVSSRYLAWYQQKPGQA QGLEWMGGIIGMFGTTDYAQKPRLLIYGASNRATGVPDRESGS FQGRVTITADEVTSTGYMELR GSGTDFTLTINRLEPEDFAVYYSLTSEDTAVYYCARGSGYHLQ CQHYSRSLTFGGGTKVEIKRT NPFDLWGRGTMQVQLQQSGAAVKKPGSSVKVS 64 QSVLTQPPSVSAAPGQMVTISC 166CKASGGRFSSYAINWVRQAPG SGSNSNIGNNYISWYQQLPGSA QGLEWMGGIIGMFGTTDYAQKPRLLIYNNYKRPSGIPDRFSAS PQGRVTITADEVTSTGYMELR KSGTSATLGITGLQTGDEADYYSLTSEDTAVYYCARGSGYHLQ CGTWDSSLSSVVFGGGTKVTVL NPFDLWGKGTLEVQLVESGGGLVQPGGSLRLS 65 QSVLTQPPSVSGAPGQRVTISC 167CAASGFPFSSYVMIWVRQVPG TGSSSNTGAGNHVHWYQQVAGA KGLEWVSAIGGSGGSTYYADSAPKLLISNNNNRPSGVPDRFSA VKGRFTISRDNSKNTLYLQMN SKSGTSASLDITGLQAEDEADYSLRADDTAVYYCVLSPKSYYD YCQSYDNSLNDWVFGGGTQLTV NSGIYFDFWGRGTL LEVQLVETGGGLVQPGGSLRLS 66 QSVVTQPPSESAAPGQKVTISC 168CAASGFPFSSYVMIWVRQVPG SGSSSNIGNNYVSWYQQFPGAA KGLEWVSAIGGSGGSTYYADSPKLLIFENNKRHSGIPDRFSGS VKGRFTISRDNSKNTLYLQMN KSGTSATLGIAELQTGDEADYYSLRADDTAVYYCVLSPKSYYD CGVWDSSLSAWVFGGGTQLTVL NSGIYFDFWGKGTLEVQLVESGGGLVQPGGSLRLS 67 NFMLTQPHSVSESPGKTVTFSC 169CAASGFPFSSYVMIWVRQVPG TRSSGSIASKYVQWYQQRPGSA KGLEWVSAIGGSGGSTYYADSPTIVIEENTKRPYGVPDRFSGS VKGRFTISRDNSKNTLYLQMN IDSSSNSASLTISGLKTEDEADSLRADDTAVYYCVLSPKSYYD YYCQSYDSSNHWVFGGGTQLTV NSGIYFDFWGRGTL LS

Use of Neutralizing Antibodies

The influenza neutralizing antibodies of the present invention can beused for the prevention and/or treatment of influenza type A infections.For therapeutic applications, the antibodies or other molecules, thedelivery of which is facilitated by using the antibodies orantibody-based transport sequences, are usually used in the form ofpharmaceutical compositions. Techniques and formulations generally maybe found in Remington's Pharmaceutical Sciences, 18th Edition, MackPublishing Co. (Easton, Pa. 1990). See also, Wang and Hanson “ParenteralFormulations of Proteins and Peptides: Stability and Stabilizers,”Journal of Parenteral Science and Technology, Technical Report No. 10,Supp. 42-2S (1988).

Antibodies are typically formulated in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The antibodies also may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization(for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively), in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules), or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,supra.

The neutralizing antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; andWO97/38731 published Oct. 23, 1997. Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated by the reverse phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257:286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et al. J National Cancer Inst. 81(19)1484 (1989).

For the prevention or treatment of disease, the appropriate dosage ofantibody will depend on the type of infection to be treated the severityand course of the disease, and whether the antibody is administered forpreventive or therapeutic purposes. The antibody is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 μg/kg toabout 15 mg/kg of antibody is a typical initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion.

The neutralizing antibodies of the present invention can be additionallyused as a tool for epitope mapping of antigenic determinants of aninfluenza A virus, and are useful in vaccine development. Indeed, asshown in the Example below, the inventors herein have identified severalbroadly reactive neutralizing antibodies that can be used as guides forvaccine design.

Thus, the neutralizing antibodies of the present invention can be usedto select peptides or polypeptides that functionally mimic theneutralization epitopes to which the antibodies bind, which, in turn,can be developed into vaccines against influenza A virus infection. Inone embodiment, the present invention provides a vaccine effectiveagainst an influenza A virus comprising a peptide or polypeptide thatfunctionally mimics a neutralization epitope bound by an antibodydescribed herein. In one embodiment, the vaccine comprises a peptide orpolypeptide functionally mimicking a neutralization epitope bound by anantibody that binds a hemagglutinin (HA) antigen. In anotherembodiments, the vaccine may be synthetic. In other embodiments, thevaccine may comprise (i) an attenuated influenza A virus, or a partthereof; or (ii) a killed influenza A virus, or part thereof. In oneother embodiment, the vaccine comprises a peptide or polypeptidefunctionally mimicking a neutralization epitope bound by an antibodythat binds a hemagglutinin (HA) antigen. The HA antigen may be an H5subtype or an H1 subtype. In another embodiment, the HA antigen isdisplayed on the surface of an influenza A virus.

In another embodiment, the peptides or polypeptides of the vaccinecontain antigenic determinants that raise influenza A virus neutralizingantibodies.

In a more general aspect, the neutralizing molecules, including but notlimited to antibodies, are useful to prevent or treat viral infections.Thus, the neutralizing molecules of the present invention are useful inboth immunotherapy, such as passive immunization using one or more suchmolecules, and in the development of vaccines directed at the viralantigenic target(s).

Identification of Residues Important for Neutralizing Function

In a significant aspect of the present invention, a cluster of antibodyresidues important for neutralizing properties have been identified. Inparticular, it has been found that antibodies comprising an antibodyheavy chain variable domain comprising at least one substitution in thesurface exposed cluster determined by amino acid positions 52A, 53, 73,and 74, following Kabat amino acid numbering, have excellentneutralizing properties, including but not limited to neutralization ofinfluenza viruses. In particular, it has been found that the followingmutations: 52A (Pro→Gly), 53 (Ile→Met), 73 (Lys→Glu), and 74 (Ser →Leuor Met), relative to germ line chemistry, create a remarkably tightcluster on the exposed surface of the 4 heavy chain variable domain,where they form a ridge that protrudes prominently from the proteinsurface. An additional mutation important for neutralizing properties,57 (Ala→Thr), is partially buried at the base of the CDR2 loop. Thesurface-exposed changes in CDR 2 and framework 3 are believed to have adirect role in antigen binding, where the less exposed mutation atposition 57 and some additional mutations are likely have indirecteffects through stabilizing and/or positioning of the CDR2 loop. Suchadditional mutations include conservative changes in CDR1 at position 34(Ile→Val) and 35 (Ser→Thr) and also in CDR2 at position 50 (Gly→Ala).These mutations are believed to be important broadly for viralneutralizing properties, including, without limitation, neutralizationof influenza A viruses, such as H5 HA, as well as HIV viruses.

These results are very valuable not only for understanding theimmunochemical basis of neutralization but also for designing antibodiesand antibody-like molecules with broad and improved viral neutralizingproperties, as disclosed and claimed herein.

Vaccine development and the development of neutralizing antibodies withimproved properties, using information about the residues important orbeneficial for neutralizing properties disclosed herein, canadditionally benefit from the combinatorial libraries ofconformationally constrained polypeptide sequence described in PCTApplication Publication No. WO 2008/089073, published on Jul. 24, 2008.

Non-Antibody Molecules with Neutralizing Properties

Although in the previous description the invention is illustrated withreference to antibody libraries, libraries of other, non-antibodymolecules, such as surrobodies, can be prepared, used, and optimized ina similar manner. Thus, the construction of unique combinatorial proteinlibraries based on the pre-B cell receptor (pre-BCR) (“surrobodylibraries”) are described in Xu et al., 2008, supra. As discussedbefore, the pre-BCR is a protein that is produced during normaldevelopment of the antibody repertoire. Unlike that of canonicalantibodies, the pre-BCR subunit is a trimer that is composed of anantibody heavy chain paired with two surrogate light chain (SLC)components. Combinatorial libraries based on these pre-BCR proteins inwhich diverse heavy chains are paired with a fixed SLC were expressed inmammalian, Escherichia coli, and phagemid systems. These librariescontain members that have nanomolar affinity for a target antigen. Adescription of the library construction, selective enrichment, andbiophysical characterization of library members is detailed in theMaterials and Methods section of the Xu et al. paper.

Further details of the invention are illustrated by the followingnon-limiting Examples.

Example 1 Antibody Libraries from Survivors of Prior Bird Flu Outbreaksand Preparation of Neutralizing Antibodies

The widespread incidence of H5N1 influenza viruses in bird populationsposes risks to human health. Even though the virus has not yet adaptedfor facile transmission between humans, it can cause severe disease andoften death. Here we report the generation of combinatorial antibodylibraries from the bone marrow of five survivors of the recent H5N1avian influenza outbreak in Turkey. To date, these libraries haveyielded >300 unique antibodies against H5N1 viral antigens. Amongstthese antibodies, we have identified several broadly reactiveneutralizing antibodies that could be used for passive immunizationagainst H5N1 virus or as guides for vaccine design. The large number ofantibodies obtained from these survivors provides a detailedimmunochemical analysis of individual human solutions to virusneutralization in the setting of an actual virulent influenza outbreak.Remarkably, two of these antibodies neutralized both H1 and H5 subtypeinfluenza viruses.

Newly emergent highly pathogenic influenza virus strains pose a profoundthreat to man. Three influenza pandemics have occurred within the past100 years, each with devastating consequences (Palese, P. & Shaw, M. L.In Fields Virology, Vol. 11 (Eds. Knipe, D M and Howley, P. M.)Lippencott Williams and Wilkins, Philadelphia, 2006, 1648-1689)). Therecent emergence of the H5N1 virus strain, though mainly confined atpresent to avian hosts, has already demonstrated virulence in humans,causing the death of more than 200 people ((2008) (World HealthOrganization, Geneva)). Therefore, healthcare officials, researchers,and governments are actively considering their options should a pandemicoccur. One widely considered approach concerns the use of passiveantibodies either for prevention of disease or treatment after exposureto virus (Luke, T. C. et al. (2006) Ann Intern Med 145, 599-609). Thepotential for passive immunization against influenza has been evidentsince the Spanish influenza nearly a century ago, where the benefits oftransfused of blood, sera, and blood products reduced the risk ofmortality by more than 50% (Id.). Recently the benefits of treatmentwith convalescent plasma have also been reported in instances of H5N1influenza (Kong L K & Zhou B P (2006) Hong Kong Med J 12, 489; Zhou, B.et al. (2007) N Engl J Med 357, 1450-1451). Additionally, passiveimmunization with human and mouse monoclonal antibodies has beenreported to protect animals from death, even when given after H5N1infection (Hanson, B. J. et al. (2006) Respir Res 7, 126)).

The most logical source of human antibodies for passive therapy would bepatients that have survived infection. Through the use of moderncombinatorial antibody library technologies, it is now possible tocapture the entire immunological history of an individual's response toan infection (Law, M. et al. (2007) Nat Med. January; 14(1):25-7. Epub2007 Dec. 6; Lerner R A (2006) Angew Chem Int Ed Engl 45, 8106-8125).Because antibody libraries contain the complete record of anindividual's response to pathogens, one can recover the repertoirespecific to a given agent by using a laboratory process of selectiveenrichment. Such libraries both give archival information as to thenature of antibodies made during the infection and allow recovery ofpotentially therapeutic human monoclonal antibodies. Importantly,antibody recovery is independent of whether an active antibody responseis still occurring at the time the sample is taken. Thus, depending onwhen the libraries are constructed, one may obtain antibodies that arecurrently being made and/or are part of the individual's immunologicalhistory. For infections that may be lethal, such analyses carried out onsurviving patients may be particularly important because they chart someof the immunological mechanisms used during a successful host defense inthe actual clinical setting of an outbreak.

Typically, when libraries are prepared from individuals who have beeninfected with a virus, hundreds to thousands of different antibodies areobtained, as opposed to only a few when other methods are used (Lerner RA (2006) Angew Chem Int Ed Engl 45, 8106-8125). This has severalconsequences. A comparative sequence analysis of these antibodies allowsa detailed map of the chemistry of antibody binding. Similarly, acomparison of neutralizing and non-neutralizing antibodies can giveimportant information about the nature of binding interactions that arecritical to neutralization.

Here we describe the creation of the first comprehensive avian influenzaantibody libraries made from survivors of infection with an avianinfluenza virus during a confirmed outbreak. We have used theselibraries to obtain large numbers of monoclonal antibodies to the H5N1avian influenza virus, some of which have broad reactivity and areneutralizing across viral sub-types. Ultimately these combinatorialantibody libraries may hold the key to immunotherapy, such as passiveimmunization, using one or more member antibodies, or they may guide thedevelopment of vaccines utilizing the antigenic target(s) of theneutralizing antibodies in the library.

The outbreak and source of material. Between December 2005 and January2006 an outbreak of avian influenza H5N1 occurred in Turkey (A. F. Oneret al., N Engl J Med 355, 2179 (Nov. 23, 2006)). In total, twelveindividuals were infected and only eight survived. Because bone marrowRNA contains the archived record of all antibodies made by anindividual, we selected it as our source material. We obtained bonemarrow and serum from six of the Turkish survivors approximately 4months following recovery and successfully prepared antibody librariesfrom five of the six bone marrow samples. In the sixth sample the RNAwas degraded.

Serological analysis. The hemagglutinin protein is essential for bindingthe influenza virus to the cell that is being infected and is generallyconsidered to be the main target of neutralizing antibodies (Palese, P.& Shaw, M. L. In Fields Virology, Vol. 11 (Eds. Knipe, D M and Howley,P. M.) Lippencott Williams and Wilkins, Philadelphia, 2006, 1648-1689)(2008) (World Health Organization, Geneva)). Therefore, we tested byELISA each of the individual serum samples at high serum dilutions forthe detection of antibodies against H5 hemagglutinin proteins (FIG. 8)and intact viruses (data not shown). This analysis showed that thepatients had readily detectable serum antibodies, even when diluted10,000-fold. We selected the Vietnam/1203/04 hemagglutinin as a targetbecause it was readily available and is thought to be related to theinfluenza virus strain that caused the outbreak of disease in Turkey.

Library construction. Our primary objectives were to understand thenature of the immunological response to infection and to identifyspecific antibodies that might be used passively for the prevention ofspread and/or treatment of H5N1 influenza virus infections. We wished torecover every possible solution to H5N1 infections with minimal or nobias. Because gene expression for the individual immunoglobulin familiesis not equal, making them prone to bias and over-representation, wedecided against using the standard pooled approach to immunoglobulinrecovery. Instead, we individually rescued 20 of the 23 distinctlyamplifiable gene families during the construction of the libraries. Theremaining three gene families (V_(H) 2, 5, and 6) were recovered as apool because they are infrequently used. We further normalized genecontent by creating equimolar pools of each immunoglobulin family DNAfor cloning into a phagemid display vector.

A unique DNA bar code was embedded into a non-disruptive portion of thephagemid vector to allow each clone to be tracked back to the originalpatient source (FIG. 9). This bar coding allows assignment of clones toindividual patients even when phage libraries from multiple survivorsare screened simultaneously. As a result of this tagging, every cloneisolated from any library can be confidently attributed to the cognatesurvivor.

As illustrated in FIG. 9, cloning and barcoding of annotated repertoiresallows tracking of all clones to their sources. Each survivor isassigned a unique barcoded vector and immunoglobulin repertoires arecloned via restriction sites indicated in the upper panel. Plasmids fromany clone can be assigned to designated sub-libraries via their lightchain class and their survivor barcode.

Using this vector with its coding system, we successfully clonedrepertoires from the bone marrow of five of the six survivors in bothsingle chain (scFv) and Fab phagemid formats. Each collection from anindividual survivor has a diversity of greater than 1.0×10⁸ members.Furthermore, we created additional bar coded libraries comprised ofmixed survivor light and heavy chains with a final diversity of 1.1×10⁹.Collectively the 5 donor-specific collections and the pooled librariesfrom all donors have a total diversity of 1.0×10⁹ as a scFv collectionand 4.2×10⁹ as a Fab-displayed collection (Table 3).

Table 3 shows the light chain and full library total transformants inboth scFv and Fab formats. Total diversity represented by all librariesis 5.6×10⁹.

TABLE 3 Light Chains Completed Libraries Kappa Lambda Kappa Lambda scFvH5-1 3.00E+06 4.00E+06 1.50E+08 1.20E+08 H5-2 3.00E+06 3.00E+06 4.00E+071.60E+07 H5-3 8.20E+05 1.70E+06 5.30E+07 1.50E+08 H5-5 7.00E+06 5.60E+066.50E+08 5.60E+07 H5-6 1.50E+06 5.00E+06 ND 1.00E+07 H5Pool NotDetermined 1.80E+08 5.70E+08 Totals 1.10E+09 9.20E+08 scFv Total2.00E+09 Fab H5-1 1.50E+06 2.90E+06 2.90E+08 4.60E+08 H5-2 3.10E+069.40E+05 4.40E+08 4.30E+08 H5-3 2.80E+06 2.30E+06 3.90E+08 3.90E+08 H5-57.00E+06 5.60E+06 7.20E+08 1.70E+08 H5-6 1.50E+06 5.00E+06 ND ND H5Pool1.90E+07 2.60E+08 Totals 2.10E+09 1.45E+09 Fab Total 3.60E+09

Selecting binding antibodies. As indicated above, one interestingfeature of these studies was that we initially selected antibodies forbinding against a virus strain and antigen that was related to, butdifferent from, the one that caused the infection. This was becauseviral isolates from the patients were not available. The necessity ofusing a related strain to select antibodies could have proven fortuitousbecause it may have led to the isolation of more broadly neutralizingantibodies (see below).

The libraries were panned against inactivated virus containing theVietnam/1203/04 virus HA and NA proteins and recombinant purifiedhemagglutinin (Barbas, C. et al. (2001) Phage Display, A LaboratoryManual (Cold Spring Harbor Laboratory Press)). Typically, followingthree to four rounds of phage panning, individual clones from enrichedphage pools were analyzed by ELISA against H5N1 virus or purifiedhemagglutinin and the positive clones were sequenced to determine theirheavy and light chain sequences and to read their survivor bar code (D.W. Coomber, Methods Mol Biol 178, 133 (2002)). From these studies, weisolated specific H5 hemagglutinin binding clones from all five of theindividual libraries from survivors. In total, we have so far recoveredmore than 300 hundred different antiviral antibodies, of which 146specifically bind the H5 hemagglutinin protein.

General Features of the Selected Clones. Overall, the individualpatients use different germ lines for both heavy and light chains,demonstrating that individual patients have found different solutions tothe same potentially lethal immunological challenge. The major featuresof combinatorial antibody libraries that can be used both to giveconfidence as to the quality of the obtained repertoire and to provideinformation as to the chemistry of antibody binding and/orneutralization are seen in these clones. These clones contain all thehallmarks of the previously described repeated clones (“jackpotsolution”) to antigen binding that is found in the natural progressionof affinity maturation, as well as in selected synthetic antibodylibraries (Lerner, R. A. Angew Chem Int Ed Engl 45, 8106 (Dec. 11,2006); A. Rajpal et al., Proc Natl Acad Sci USA 102, 8466 (Jun. 14,2005)). The presence of “jackpots” in these large collections validatesthe screening procedure because, unless the phage were selected on thebasis of activity, the chance of obtaining the same clone multiple timesis highly improbable. Moreover, when one analyzes the heavy chaindifferences within groups, it was observed that many of the amino acidsubstitutions were chemically and structurally conservative (Table 1).As with repeated clones, the appearance of multiple amino acidsubstitutions that are chemically reasonable is unlikely to be a randomevent.

Binding Specificity of recovered antibodies. Initial testing of a set ofFabs using Bio-Layer Interferometry binding to the H5 Vietnamhemagglutinin protein indicated that we had identified at least fourdistinct epitopes (data not shown). We selected six clones from threesurvivors that recognized two different epitopes for conversion intofull IgG, proteins. The binding of three of these antibodies was mappedto the HA1 subunit of the hemagglutinin protein by western blot analysis(data not shown).

One goal of these studies was to recover those rare antibodies thatbroadly neutralize divergent viral strains. There was a suggestion thatsome of our antibodies might be broadly reactive because the serum fromthe donors had high titer antibodies against a divergent subfamily ofH5N1 viruses that extended beyond the virus with which they wereinfected. To determine the degree of cross reactivity at the level ofindividual antibodies, we analyzed binding of our clones to differentinfluenza hemagglutinin antigens (FIG. 6).

FIG. 6 shows the cross-reactivity of H5N1 antibodies from two survivorswith hemagglutinins from H1N1 viruses. (A) Bars are H5N1 Vietnam 1203/04(dark grey), H5N1 Turkey/65596/06 (white), H5N1 Indonesia/5/05 (diagonalstripes), H1N1 New Calcdonia/20/99 (vertical stripes), H1N1 SouthCarolina/1/18 (crosshatch stripes), and H3N2 Wisconsin/67/05 (lightgray). (B) Relative ranking of antibodies by their ELISA signal overbackground (“+” is above background and less than 2 fold, “++” isbetween 2 and 9-fold, “+++” is between 9 and 15-fold, “++++” is greaterthan 15-fold above background, and “−” is not measurably abovebackground), on the various proteins. Not surprisingly, these antibodiesrecognize hemagglutinin from the corresponding infecting Turkey/65596/06strain, and in addition recognize the heterologous hemagglutinin fromthe Vietnam/1203/04 strain used for selection. Furthermore, theyrecognize the antigenically divergent Indonesian/5/05H5 hemagglutinin.We performed kinetic binding analyses on prototype antibodies and foundthat the antibodies from survivor 5 bound Vietnam/1203/04 hemagglutininwith single digit nanomolar affinities, while the survivor 2 antibodybound more strongly with a measured affinity of 13 μM. The availablebinding affinities for H5 and H1 and the clonal identities of the Fabfragments for the antibodies are as follows.

Identity H5 H1 A 10-40 nM 40-80 nM B 130 pM C 10-40 nM D 13 pM E 4 nM 90nM

To determine if our antibodies are even more broadly reactive, westudied their binding to a larger collection of hemagglutinins fromdifferent influenza A subtypes (FIG. 6). We found that the fourprototype antibodies bound hemagglutinin from the closely relatedsubtype H1N1 contemporary reference strain New Calcdonia/20/99. Notably,the three neutralizing antibodies belonging to survivor 5 also boundhemagglutinin from the H1N1 South Carolina/1/18 isolate that emergedduring the 1918 Spanish Flu pandemic. Conversely, none of these fourantibodies bound hemagglutinin from the contemporary H3N2Wisconsin/67/05 reference strain, indicating that even though theantibodies display broad spectrum binding amongst and between Influenzasubtypes, the reactivity did not extend to all influenza subtypes.

To further explore the immunochemical basis of the H1/H5cross-reactivity, we re-screened the libraries against the H1N1 NewCalcdonia/20/99 hemagglutinin protein. From this selection, we foundclones (Table 6) that bore significant similarity to the sequencespreviously obtained from survivor 5 when the H5 hemagglutinin proteinwas used in the panning (Table 5).

Neutralization Studies. Initially the antibodies were assayed for theirability to neutralize an H5 HA (Vietnam/1203/04) containing influenzavirus. One antibody derived from survivor 2 and 3 from survivor 5 thatrecognized a common epitope (epitope “A”) were all neutralizing whereasthe two antibodies derived from survivor 1 that recognized a secondepitope (epitope “B”) were not.

Based on the striking sequence similarity of clones separately isolatedfrom survivor 5 against either H5N1 or H1N1 hemagglutinin, we predictedthat their cross reactivity would extend beyond simple binding and theywould also have the highly unusual property of neutralizing both H5N1and H1N1 virus. To test the cross neutralizing activity of the IgGs, wetested representative antibodies from the H5N1 screen in aneutralization assay to see if they would also neutralize H1N1 or H3N2virus (Table 4). We studied the H1 bearing virus A/New Cal/20/99 and theH3 bearing virus A/Hong Kong/68. A collection of viruses bearing H5subtype hemagglutinin was also tested (A/Vn/1203/04; A/Indo/5/05;A/Turkey/65596/06; A/Egypt/06). The antibodies showed no activityagainst H3 subtype influenza. However, three of the monoclonalantibodies (1-3) that neutralized H5 containing viruses also stronglyneutralized all viruses bearing HA from subtypes H1 (Table 4).

TABLE 4 Virus* H5 H1 H3 A/Vietnam A/Vietnam A/Indonesia A/Turkey A/EgyptA/New Cal A/Hong 1203/04 ‡ 1203/04 ‡ 5/05 65596/06 14725/06 20/99Kong/68 Ab 1† 11-21 2.3-9.3 9.3 9.3 1.2-2.3 9 >333 Ab 2† 63  54-217 27108  7-13 54-108 >333 Ab 3† 58 18 16 31 4-8 8-16 >333 Ab 4† 1.7-6.30.5-2.2 >333 Not done Not done >333 >333 Mab #8§ 2.7 Not done Not doneNot done Not done Not done Not done The results of Table 4 were obtainedas follows. MDCK cells were inoculated with 100 TCID50 of virus in thepresence of 2-fold serial dilutions of monoclonal antibodies. †Minimuminhibitory concentrations required to neutralize virus in duplicatesamples are presented in ug/ml. ‡ - The viral neutralization resultsfrom two independent experiments are both shown. §Mab#8 is a mousemonoclonal H5N1 neutralizing antibody raised against A/Vietnam/1203/04.

The results of Table 4 were obtained as follows. MDCK cells wereinoculated with 100 TCID50 of virus in the presence of 2-fold serialdilutions of monoclonal antibodies. †—Minimum inhibitory concentrationsrequired to neutralize virus in duplicate samples are presented inug/ml. ‡—The viral neutralization results from two independentexperiments are both shown. §—Mab#8 is a mouse monoclonal H5N1neutralizing antibody raised against A/Vietnam/1203/04.

Immunochemical basis of neutralization. One advantage of antibodylibraries is that when one obtains large numbers of antibodies, they canbe grouped as to their relatedness. Thus, when a function for a givenantibody in the collection is observed one can predict that othermembers of the group to which it belongs will have similar activity.

Table 5 shows example sequences displaying the immunochemical basis ofneutralization discovered from Survivor 5 libraries following H5N1Vietnam panning. The 61 unique heavy chain sequences aligned with theirgermline variable regions (variable (V) region gene V_(H)1e/V_(H)1-69)from the 114 unique heavy and light chain combinations. Requisitemutations are shown in bolded, underlined text (column 5—PI to GM and Ato T; column 6—KS to EL or EM or XL) and predominant mutations are shownin italicized, underlined text (column 2—A to T; column 3—IS to VT;column 5—G to A; column 8—K to Q or R). Heavy chains sequences alsodiscovered in H1N1 New Calcdonia panning are highlighted in gray.Antibody regions and Kabat numbering ranges are listed at the top ofeach sequence column. The heavy chain/light chain pairing is indicatedin the first column as follows: *—paired with 2 unique light chains,†—paired with 3 unique light chains, ‡—paired with 4 unique lightchains, §—paired with 5 unique light chains, and ¶—paired with 12 uniquelight chains.

TABLE 5 Group 1 heavy FR1 CDR1 FR2 CDR2 chains 1-29 30-35 36-46 47-56Vh1e QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYAIS WVRQAPGQGLE WMGGIIPIFGTAN 1----------------------------- ------ ----------- ------ GM ---T- 2----------------------------- ------ ----------- --- A -- GM ---T- 3----------------------------- ------ ----------- --- A -- GM ---T- 4*----------------------------- ---- VT ----------- --- A -- GM ---T- 5----------------------------- ---- VT ----------- --- A -- GM ---T- 6----------------------------- ---- VT ----------- --- A -- GM ---T- 7*----------------------- T ----- ---- VT ----------- ------ GM ---T- 8*----------------------- T ----- ---- VT ----------- ------ GM ---T- 9†----------------------- T ----- ---- VT ----------- --- A -- GM ---T- 10----------------------- T ----- ---- VT ----------- --- A -- GM ---T-11* ----------------------- T ----- ---- VT ----------- --- A -- GM---T- 12¶ ----------------------- T ----- ---- VT ----------- --- A --GM ---T- 13* ----------------------- T ----- ---- VT ----------- --- A-- GM ---T- 14 ----------------------- T ----- ---- VT ----------- --- A-- GM ---T- 15 ----------------------- T ----- ---- VT ----------- --- A-- GM ---T- 16* ----------------------- T ----- ---- VT ----------- ---A -- GM ---T- 17 -----------------R----- T ----- ---- VT ----------------- GM ---T- 18 ----------------------- T ---S- ---- VT ----------------- GM ---T- 19 ----------------------- T T---- ---- VT ----------------- GM ---T- 20§ ----------------------- T T---- ---- VT ----------------- GM ---T- 21 ----------------------- T T---- ---- VT ----------------- GM ---T- 22 ----------------------- T T---- ---- VT ----------------- GM ---T- 23 -----------------------X----- ---- VT ----------- ---A -- GM ---T- 24 E---------------------------- ------ ----------- --- A-- GM ---T- 25 E-------T----------L--------- ---- VT ----------- ------GM ---T- 26 E-------T--------------V----- ---- VT ----------- --- A --GM ---T- 27 E----------Q----------- T ----- ---- VT ----------- --- A --GM ---T- 28 E--------------A-----------A- ---- VT ----------- ------ GM---T- 29† E-----------------R---- T ----- ---- VT ----------- ------ GM---T- 30† E-----------------R---- T ----- ---- VT ----------- ------ GM---T- 31 E-----------------R---- T ----- ---- VT ----------- ------ GM---T- 32 E---------------------- T ----- ---- VT ----------- --- A -- GM---T- 33* E---------------------- T ----- ---- VT ----------- --- A --GM ---T- 34‡ E---------------------- T ----- ---- VT ----------- --- A-- GM ---T- 35 E---------------------- T ----- ---- VT ----------- --- A-- GM ---T- 36 E---------------------- T ----- ---- VT ----------- --- A-- GM ---T- 37 E---------------------- T ----- ---- VT ----------- --- A-- GM ---T- 38 E---------------------- T ----- ---- VT ----------- --- A-- GM ---T- 39§ E---------------------- T ----- ---- VT ----------- ---A -- GM ---T- 40§ E---------------------- T ----- ---- VT -------------- A -- GM ---T- 41* E---------------------- T ----- ---- VT----------- --- A -- GM ---T- 42† E---------------------- T ----- ----VT ----------- --- A -- GM ---T- 43 E---------------------- T ----- ----VT ----------- --- A -- GM ---T- 44 E---------------------- T ----- ----VT ----------- --- A -- GM ---T- 45 E---------------------- T ----- ----VT ----------- ------ GM ---T- 46 E---------------------- T ----- ----VT ----------- ------ GM ---T- 47 E---------------------- T ----- ----VT ----------- ------ GM ---T- 48† E---------------------- T ----- ----VT ----------- ------ GM ---T- 49 E---------------------- T ----- ----VT ----------- ------ GM ---T- 50† E---------------------- T T---- ----VT ----------- ------ GM ---T- 51 E---------------------- T T---- ----VT ----------- ------ GM ---T- 52 G---------------------- T ----- ----VT ----------- --- A -- GM ---T- 53 G---------------------- T ----- ----VT ----------- --- A -- GM ---T- 54 -M--------------------- T ----- ----VT ----------- --- A -- GM ---T- 55† -M--------------------- T --------- VT ----------- --- A -- GM ---T- 56 ----L------------------ T T-------- VT ----------- ------ GM ---T- 57* ----Q------------------ T --------- VT ----------- --- A -- GM ---T- 58 ----Q------------------ T --------- VT ----------- --- A -- GM ---T- 59 ----Q------------------ T --------- VT ----------- ------ GM ---T- 60* ----Q-------------R---- T --------- VT ----------- ------ GM ---T- 61* ----Q-------------R---- T --------- VT ----------- ------ GM ---T- Group 1 heavy FR3 CDR3 FR4 chains59-92 93-101 102-113 Vh1e YAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGSYYYESSLD YWGQGTLVTVSS 1 --------------EL--------------------------------- --- K -T----- 2 ----------L---EL--------------------------------- --- K -M----- 3 --------------EL-------------D------------------- --- K -M----- 4* ----------L---EL--------------------------------- ------------ 5 --------------EL-------------D------------------- ------------ 6 ----------L---EL--------------------------------- --- K -M----- 7* --------------ELR-------------------------------- ------------ 8* --------------EL--------------------------------- --- R ------- 9† --------------EL-------------D------------------- ------------ 10 --------------EL-------------D------------------- --- R -M----- 11* --------------EL-------------D------------------- ------M----- 12¶ ----------L---EL--------------------------------- ------------ 13* ----------L---EL--------------------------------- ------M----- 14 ----------L---EL--------------------------------- --- K -M----- 15 ----------L---EL-------------X------------------- ------------ 16* ----------L---EL--------------------------------- ------------ 17 --------L-----EM-----------------------------TT-- --- R ------- 18 --------------EL--------------------------------- ------M----- 19 --------------EM--------------------------------- --- K -M----- 20§ --------------EM--------------------------------- ------------ 21 --------------EM--------------------------------- --- K ------- 22 --------------EM--------------------------------- ------M----- 23 --------------EL--------------------------------- ------------ 24 --------------EL--------------------------------- ------------ 25 --------L-----EM--------------------------------- --- R -M----- 26 --------------EL-------------D---------------TT-- ---G--T----- 27 ----------L---EL--------------------------------- ------------ 28 --------------EL--------------------------------- ------M----- 29† --------L-----EM--------------------------------- ------------ 30† --------L-----EM-----------------------------TT-- --- K ------- 31 --------L-----EM-----------------------------TT-- --- R -M----- 32 ----------L---EL-----------------------------TT-- --- R ------- 33* --------------EL-------------D------------------- --- K ------- 34‡ --------------EL-------------D------------------- ------------ 35 --------------EL-------------D------------------- ------M----- 36 --------------EL-------------D------------------- --- K ------- 37 --------------EL-------------D------------------- --- K -T----- 38 --------------EL-------------D------------------- --- K -M----- 39§ ----------L---EL--------------------------------- ------------ 40§ ----------L---EL--------------------------------- --- K ------- 41* ----------L---EL--------------------------------- --- K -M----- 42† ----------L---EL--------------------------------- --- R ------- 43 ----------L---XL--------------------------------- --- R ------- 44 ----------L---EL--------------------------------- --- R -M----- 45 ----------M---EM--------------------------------- --- K ------- 46 --------------EL--------------------------------- ------------ 47 --------------EL--------------------------------- --- K ------- 48† --------------EM--------------------------------- --- R ------- 49 --------------EM--------------------------------- --- R -M----- 50† --------------EM--------------------------------- ------------ 51 --------------EM--------------------------------- ------M----- 52 ----------L---EL--------------------------------- --- K -M----- 53 --------------EL-------------D------------------- --- R ------- 54 --------------EL-------------D------------------- --- K -M----- 55† ----------L---EL--------------------------------- ------------ 56 --------------EM--------------------------------- --- K ------- 57* ----------L---EL--------------------------------- ------------ 58 --------------EL-------------D------------------- --- K -M----- 59 --------------EL-----------------------------N--- --- K ------- 60* --------L-----EM-----------------------------TT-- ------M----- 61* --------L-----EM------------------------------TT- --- K -------

All members of the group that contained the neutralizing antibodycollection against epitope “A” from survivor 5, analyzed to date, areshown in Table 1. The group is comprised of 61 unique members that mostclosely resemble the variable (V) region gene V_(H)1e/V_(H)1-69 germline heavy chain. Some heavy chains are paired with more than one lightchain. In total these heavy chains have 114 unique pairings to bothkappa and lambda light chains. Comparing these heavy chains to thehighly related V_(H)1e/V_(H)1-69 germline, we observe three types ofpoint substitutions. Some changes appear to be required, others aredominant, and some residues have only been changed sporadically. Thechanges that are required occur in every clone in the group within CDR2at position 52A (Pro>Gly), 53 (Ile>Met), and 57 (Ala>Thr), as well as inthe framework 3 region at position 73 (Lys>Glu) and 74 (Ser>Leu or Met),all of which vary from the germline side chain chemistries, suggestingthat these mutations are critical to antigen binding and neutralization.The second set of mutations is dominant and found in most clones. Thefirst, in framework 1 at position 24 (Ala>Thr), represents a significantchemical change. The next three are conservative changes in CDR1 atpositions 34 (Ile>Val) and 35 (Ser>Thr) and also in CDR2 at position 50(Gly>Ala). All four of these dominant substitutions, however, aredispensable, suggesting that, while beneficial, they are not essential.The sporadic changes found throughout framework regions 1, 3, and 4, aswell as CDR3, are all conservative and likely represent minoroptimization events.

Table 6 shows examples of sequences displaying the Immunochemical basisof neutralization discovered from Survivor 5 libraries following H1N1New Calcdonia panning. The 35 unique heavy chain sequences aligned withtheir germline variable regions from the 82 unique heavy and light chaincombinations. Requisite mutations are highlighted in bolded, underlinedtext (column 2—A to T; column 3—IS to VT; column 5—PI to GM and A to T;column 6—KS to EL or EM and K to E) and predominant mutations are shownin italicized, underlined text (column 2—Q to E; column 5—G to A; column6—I to L or M; column 8—Q to K or R or E). Heavy chains sequences alsodiscovered in H5N1 Vietnam panning are highlighted in gray.

TABLE 6 Group 1 heavy FR1 CDR1 FR2 CDR2 chains 1-2930-35 30-40 47-56Vh1e QVQLVQSGAEVKKPGSSVKVSCKASGGTF SSYAIS WVRQAPGQGLE WMGGTIPIFGTAN 1*     VT    A   GM    T 2‡                        T      VT        GM   T 3 -----------------------T----- ---- VT ----------- ------ GM ---T-4 -----------------------T----- ---- VT ----------- ------ GM ---T- 5-----------------------T----- ---- VT ----------- ------ GM ---T- 6†                       T      VT    A   GM    T 7-----------------------T----- ---- VT ----------- ------ GM ---T- 8*                       T      VT    A   GM    T 9¶                       T      VT    A   GM    T 10*                       T      VT    A   GM    T 11-----------------------T----- ---- VT ----------- ------ GM ---T- 12-----------------------T----- ---- VT ----------- ------ GM ---T- 13-----------------------T----- ---- VT ----------- ------ GM ---T- 14-----------------------T----- ---- VT ----------- ------ GM ---T- 15-----------------------T----- ---- VT ----------- ------ GM ---T- 16§                       TT      VT        GM    T 17-----------------------TT---- ---- VT ----------- ------ GM ---T- 18E--------------A-------T----- ---- VT ----------- ------ GM ---T- 19†E                 R    T      VT        GM    T 20‡E                      T      VT    A   GM    T 21E                      T      VT    A   GM    T 22E                      T      VT    A   GM    T 23§E                      T      VT    A   GM    T 24§E                      T      VT    A   GM    T 25*E                      T      VT    A   GM    T 26*E----------------------T----- ---- VT ----------- ------ GM ---T- 27†E                      T      VT    A   GM    T 28E----------------------T----- ---- VT ----------- ------ GM ---T- 29†E                      T      VT        GM    T 30E----------------------TT---- ---- VT ----------- ------ GM ---T- 31E----------------------TT---- ---- VT ----------- ------ GM ---T- 32† M                     T      VT    A   GM    T 33----E------------------T----- ---- VT ----------- ------ GM ---T- 34*    Q                  T      VT    A   GM    T 35*    Q             R    T      VT        GM    T Group 1 heavy FR3 CDR3FR4 chains 59-92 93-101 102-113 Vh1eYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYC ARGSYYYESSLD YWGQGTLVTVSS 1*           L     EL 2‡                EL R 3 ----------M--- EM--------------------- ------------ ------------ 4 ----------M--- EM--------------------- ------------ ------M----- 5 -------------- EM--------------------- ------------ ------------ 6†                EL             D 7 -------------- EL -------------D------- --------------- K -------- 8*                EL              D       M 9¶           L     EL 10*            L     EL       M 11 ---------- L --- EL--------------------- ------------ ------P----- 12 ---------- L --- EL--------------------- ------------ --- R -------- 13 ---------- L --- EL--------------------- ------------ --- R --M----- 14 ---------- L --- EL--------------------- ------------ --- K -------- 15--------------E---------------------- --------T--- --- K -------- 16§               EM 17 -------------- EM --------------------------------- --- R --M----- 18 -------------- EM --------------------------------- ------------ 19† --------L----- EM ---------------------        TT     K 20‡                EL              D 21               EL              D        M 22                EL              D      K23§            L     EL 24§            L     EL      K 25*            L    EL      K   M 26* ---------- L --- EL --------------------------------- --- K --T----- 27†            L     EL      R 28-------------- EL --------------------- --------N--- --- R -------- 29†               EM      R 30 -------------- EM --------------------------------- --- R --M----- 31 -------------- EM --------------------------------- --- K --M----- 32†            L     EL 33 ---------- L ---EL --------------------- ------------ ---E--T----- 34*            L    EL 35*        L       EM         TT       M

In Table 6, antibody regions and Kabat numbering ranges are listed atthe top of each sequence column. (*—paired with 2 unique light chains,^(†)—paired with 3 unique light chains, ^(‡)—paired with 4 unique lightchains, ^(§)—paired with 5 unique light chains, ^(‡)—paired with 13unique light chains).

FIG. 7 shows the positions of the required mutations in the structure ofthe antibody superimposed on the crystal structure of a highly relatedanti-HIV Fab called 47e (1rzi.pdb) (Huang, C. C. et al. (2004) Proc.Nat. Acad. Sci. 101, 2706-2711). FIG. 7 shows the positions of H5hemagglutinin binding Group 1 required and dominant mutations on thecrystal structure of Fab 47e. The required mutations are shown as G52(52A (Pro>Gly)), M53 (Ile>Met), T57 (Ala>Thr), E73 (Lys>Glu) and LM74(Ser>Leu or Met). The dominant mutations are shown as T24 (Ala>Thr), V34(Ile>Val), T35 (Ser>Thr), and A50 (Gly>Ala). The required and dominantGroup1 heavy chain sequences identified in H5 Vietnam/1203/2004 HAbiopanning are superimposed on the crystal structure of the highlyrelated anti-HIV Fab 47e. Mutations are shown in both backbone (top) andspace-filling (bottom) models. A tight cluster is formed by four of therequired mutations in and adjacent to CDR2. The required mutations 52A(Pro>Gly), 53 (Ile>Met), 73 (Lys>Glu) and 74 (Ser>Leu or Met) form aremarkably tight cluster on the exposed surface of the heavy chainvariable domain where they form a ridge that prominently protrudes fromthe protein surface (FIG. 7). The remaining required mutation 57(Ala>Thr) is partially buried at the base of the CDR2 loop. The surfaceexposed changes in CDR 2 and framework 3 are likely to have a directrole in antigen binding while the less exposed required mutation and thenon-essential dominant mutations may have indirect effects throughstabilizing and/or positioning the CDR2 loop.

The antibodies from survivor 2 are comprised of 2 unique heavy chainsthat most closely resemble the V_(H)4-4b germ line heavy chain (Table7). The first heavy chain has been found paired with 5 unique lambdalight chains, four of which are from the infrequently used lambda 6light chain family and the other is paired with a single kappa lightchain. Antibody 4 whose neutralization profile was more restricted camefrom this group.

TABLE 7 FR1 CDR1 FR2 CDR2 Group 2 (1-29) (30-35)(36-46) (47-58) HeavyQVQLQESGPGLVKPSETLSLTCTVSGYSF DSGYYWG WLRQPPGKGLE WIGSIYHSRNTY chainLambda FMLTQPHSVSESPGKTVTISCTGSGGN IARNYVQWY QQRPGSAPV TVILEDDKRP lightFMLTQPHSVSESPGKTVTISCTGSSGS IASNYVQWY QQRPGSAPT TVIYEDYQRP chainsSVLTQFPSASGTPGQRVTISCSGSSSN IGSNTVNWY KQLPGTAPR LLIYSNDQRPSVLTQPPSASGTPGQRVTLSCSGSSSN IGGNSVNWY QHVPGTAPK LLMHSDDQRPPELTQPHSVSESPGKTVTISCTGSGGR IATNHVQWY QQRPGSAPT IVIYENNQRPPELTQPPSASGTPCQRVTISCSGSSSN IGSNTVNWY QQLPGTAPK LLIYSNNQRP KappaDIQMTQSPSSLSAFVGDRVTITCQASQDI SNYLNWY QQKPGKAPK LLIYDATNLE light chainFR3 CDR3 FR4 Group 2 (59-92) (93-101) (102-113) HeavyYNPSLKSRVTISVDTSKNQFSLQLSSVTAADTAVYYC ARGTWYSSNLRYWFD PWGKGTLVRVSS chainLambda SGIPDRFSGSIDRSSNSASLTISGLRTEDEALYYC QSYDDSDLV VFGGCTKLT lightSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYC QSYDDSDHL IFGGGTKLTVL chainsSGVPDRFSGSKSGTSASLAISGLQSEDEANYYC AAWDDSLSGW VFGGGTKLTVLSGVPDRFSGSKSGTSXSLAISGLQSEDEADYYC AXWDDSLNAW VFGGXTKVTVLSGVPNRFSGSIDDSSNSASLTTSALRTEDEADYYC QSADATNV FFGGGTKVTVLSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGW VFGGXTKLTVL KappaTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPL TFGGGTKVDIKR light chain

The probability that a given mutation is important to the activity of anantibody increases as a function of the number of times it wasindependently selected. To determine if the required mutations wereselected during somatic mutation from independent clones or were fromthe progeny of a single clone that further mutated during subsequentreplications, the codon usage of the dominant mutations were analyzed(Table 8A-8B). The data reveal that although different codons were usedthey resulted in the same amino acid changes, demonstrating that thesemutations arose independently in different clones and were, thus,selected multiple times. This convergent outcome for independentlyselected events is strong evidence that these dominant mutations play acritical role in the binding to the virus and/or its neutralization.

As illustrated in Table 8A-8B, codon usage of individual clones showsindependent origin of selected H5 HA binding clones. DNA alignment andencoded amino acids for 6 representative Group 1 antibodies against theVH1-e germline. The use of different codons for the same amino acidsdemonstrates that each unique sequence is of a distinct origin. Table 8Acorresponds to CDR2 and Table 8B corresponds to Framework 3. Germ linecodons are shown as bolded codons. A change from a germ line codon tothe same amino acid is shown as a plain text codon. A first change froma germ line amino acid is shown as a bolded, underlined codon. A secondchange from a germ line amino acid is shown as an italicized, underlinedcodon. A third change from a germ line amino acid is shown as anunderlined, grayed-out codon.

TABLE 8A Kabat Sequence CDR2 Clone Number 47 48 49 50 51 52 52A 52B 52053 54 55 56 57 58 VH1-e germilne TGG ATG GGA AGG ATC ATC CCT ATC CTT GGTATA GCA AAC TAC GCA W M G R I I P I L G I A N Y A 27 TGG ATG GGC GCG ATCATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W M G A I I G M F G T T N Y A 30TGG ATG GGA GGG ATC ATC GGT ATG TTT GGA ACA ACC AAC TAT GCA W M G G I IG M F G T T N Y A 33 TGG ATG GGA GCG ATC ATC GGT ATG TTT GGT ACA ACA AACTAC GCA W M G A I I G M F G T T N Y A 41 TGG ATG GGC GCG ATC ATC GGT ATGTTT GGT ACA ACA AAC TAC GCA W M G A I I G M F G T T N Y A 50 TGG ATG GGAGGG ATC ATC GGT ATG TTT GGT ACA ACG AAC TAT GCA W M G G I I G M F G T TN Y A 17 TGG ATG GGA GGG ATC ATC GGT ATG TTT GGT ACA ACA AAC TAC GCA W MG G I I G M F G T T N Y A #Codons used 1 1 2 2 1 1 1 1 1 2 1 3 1 2 1

TABLE 8B Kabat Sequence Clone Framework 3 Number 67 68 69 70 71 72 73 7475 76 77 78 VH1-e GTC ACG ATT ACC GCG GAC AAA TCC ACG AGC ACA GCCgermline V T I T A D K S T S T A 27 GTC ACG CTT ACC GCG GAC GAA TTA ACGTCC ACA GCC V T L T A D E L T S T A 30 CTC ACA ATC ACC GCG GAC GAG ATGACG TCC ACA GCC L T I T A D E M T S T A 33 GTC ACA ATC ACC GCG GAC GAATTA ACG TCC ACA GCC V T I T A D E L T S T A 41 GTC ACG CTT ACC GCG GACGAA TTA ACG TCC ACA GCC V T L T A D E L T S T A 50 GTC ACG ATT ACC GCGGAC GAG ATG ACG TCC ACA GCC V T I T A D E M T S T A 17 GTC ACG ATT ACCGCG GAC GAA TTA ACG TCC ACA GCC V T I T A D E L T S T A #Codons used 2 23 1 1 1 2 2 1 1 1 1

The present report raises two central issues relative to the preventionand treatment of infections caused by the avian influenza neutralizedvirus. The first concerns the importance of antibodies relative to othercomponents of the immune system. While it has been known for over 80years that passive administration of immune sera can prevent infectionLuke, T. C. et al., Kilbane E M, Jackson J L, & Hoffman S L (2006) AnnIntern Med 145, 599-609), more recent studies with monoclonal antibodiesalso offer encouragement (Hanson, B. J. et al. (2006) Respir Res 7, 126;Huang, C. C. et al. (2004) Proc. Nat. Acad. Sci. 101, 2706-2711; SimmonsC. P. et al. (2007) PLoS Med 4, e178). For example, Hanson et. al.showed that a monoclonal antibody to H5N1 virus was completelyprotective against lethal infection, even when given three days postinoculation in mice (Hanson, B. J. et al. (2006) Respir Res 7, 126).Given the possibility of a catastrophic epidemic, the way forward seemsclear to many in the field. It has been suggested that governmentsshould maintain stocks of neutralizing antibodies such as those reportedhere. The facts that our antibodies are fully human and have beenisolated from individuals who successfully combated viral infection mayoffer advantages. However, even if such antibodies are stockpiled,hurdles remain. For instance, if the gene encoding the epitope to whichthe antibody binds were to mutate, then the antibody might be lesseffective. Also, there is some evidence that cellular immunity enhancesclearance of the virus. Nevertheless, if the only effect of passiveimmunization was to lessen the severity of infection, thereby giving thenecessary time for other immune effectors to operate, it could be ofcritical importance for lessening mortality in patients with weakenedimmune, cardiovascular, and respiratory systems and in the elderly.Passive immunization might prevent the cytokine storm against rapidlyproliferating virus, as occurred even in healthy young adults during the1918 influenza outbreak.

The second important feature of this report relates to the specialadvantages that antibodies from combinatorial libraries bring to theproblem (Lerner R A (2006) Angew Chem Int Ed Engl 45, 8106-8125). Themost general aspect is that, because such libraries are nucleic acidbased, they are not are not dependant on whether an important antibodyis currently being produced. This obviates any concern about when in thecourse of the disease the sample was obtained. Indeed, as is the casehere, when the source of antibody genes is the bone marrow, the entireimmunologic history of an individual's antibody response may beobtained, irrespective of whether an antibody is actively expressed oris stored in the memory compartment. Thus, in the analysis of antibodyontogeny in the individuals studied here, the time factor is eliminatedand one can get a clearer view of the precursor product relationshipsbetween related antibodies. In this respect, one of the most remarkablefeatures of some of our antibody collections (i.e. group 1) is that therequired somatic mutations are confined to framework 3 or CDRH2 ratherthan CDRH3 where they would be most expected to occur. This may bebecause the extreme virulence of the virus imposes time pressure on theevolution of the immune response. To survive an H5N1 avian influenzavirus infection, one must mount an effective immune response rapidly.Because the framework regions and CDR2 of the protein are structurallyrather constrained, the evolutionary search of sequence space forincreased binding energy through somatic mutation may be more efficientfor these regions than for a similar search through the more flexibleand diverse CDR3 region. Indeed, it is well known, mostly from attemptsto humanize antibodies, that framework mutations can directly orindirectly affect binding energy and/or specificity (Foote J & Winter G,(1992) J Mol Biol 224, 487-499; Holmes, M. A. et al. (2001) J Immunol167, 296-301). Alternatively, the immune system may use frameworksand/or CDRs that have been previously optimized, perhaps in response toan earlier exposure to a similar virus. Regardless of the exactmechanism, our results are in broad agreement with those of Zinkernageland colleagues who studied the immune response against lethal vesicularstomatitis virus infections in mice (Kalinke U et al. (1996) Immunity 5,639-652; Kalinke U et al., (2000) Proc Natl Acad Sci USA 97,10126-10131). In their studies, only one V_(H) germline gene was usedand the primary neutralizing immune response was devoid of somaticmutations. Only later did somatic mutations in the CDRs appear. Itshould be emphasized that while our analysis to date has revealed manyinteresting antibodies, we have so far only analyzed a small fraction ofthe library. As further analyses are carried out, we expect to see manyother immunochemical solutions to the problem of virus infection.

From an antibody engineering point of view, the large database unique toantibody libraries creates a roadmap for improving the binding energyand/or specificity of the antibodies, if necessary. For example, oneunderstands immediately that there are heavy chains (Tables 1, 2, andS14) that are highly promiscuous with respect to their light chainpartners. These heavy chains are ideal for light chain shufflingexperiments where very large numbers of new light chains are paired witha single promiscuous heavy chain (Lerner R A (2006) Angew Chem Int EdEngl 45, 8106-8125; Kang, A. S. et al. (1991) Proc Natl Acad Sci USA 88,11120-11123). Ultimately, the best features of different antibodies canbe amalgamated into a single antibody that can be highly effective andeven overcome viral escape by mutation. This is especially likely whenconsensus sequences important to neutralization occur either in thedifferent antibody chains or in different CDRs or frameworks within achain or both. Thus, many combinations can be tested and an amalgamatedantibody could contain the best elements of these various loops andframeworks. Critically, when some of the features incorporated into theamalgamated antibodies represent alternative binding modes to aneutralization target on the virus, one would expect viral escape to bemore difficult.

There is another feature that derives from the large numbers ofantibodies obtained from libraries that may be of particular importanceto the influenza problem. Many, if not most, of the antibodies thatresult from an infection have little to do with prevention of furtherinfectivity and are simply a response to the foreign nature of thevirus. Thus, if one has only a few antibodies to choose from, one mightmiss the most important rare antibodies because they areunder-represented in the bulk immune response. Indeed, this may be afeature of the most potent antibodies since they need only be present insmall concentrations and/or may occur late in an infection only aftermany other “attempts” were tried during the evolution of an immuneresponse. We have seen this phenomenon in human libraries from cancerpatients where antibodies that prevent metastasis are present at thevery rare frequency of about one out of 1.0×10⁸ library members(Felding-Habermann B, et al. (2004) Proc Natl Acad Sci USA 101,17210-17215). The features that one might screen for that would beexpected to be rare are, for example, antibodies that exhibit broadneutralization or have unusual access to important tissue compartments.Toward this end, it will be interesting to see if there are anyneutralizing antibodies in our collection of clones that bind virus butare not directed to the hemagglutinin.

The analysis of the immune response from actual cases can give guidancefor both new passive antibody therapy and vaccine design. For example,we already know that patients make antibodies against the hemagglutininthat are broadly reactive between H5 and H1 strains, but skip H3. Wecould not learn this from simple serology because serum contains acollection of activities as seen here for our patients and, thus, it isimpossible to determine the clonal basis of any reactivity from ananalysis of sera. The localization of the cross-reactive epitopesalready found here as well as others is now relatively straight forwardusing antibodies from the library as a guide. Access to multipleantibodies from several survivors of the viral infection also enablesthe mapping of common epitopes, other than hemagglutinin, to which allsurvivors have developed high affinity antibodies. The knowledge ofseveral previously unknown epitopes could provide the foundation for thedesign of novel vaccines.

Characterized neutralizing antibodies can also give informationregarding the potential efficacy of candidate vaccines. For instance,one can determine if particular traditional or recombinant vaccinepreparations generate antibody classes that have proven to beneutralizing from analysis of survivors of actual infections.Furthermore, these antibodies can be used as test reagents to ensurethat epitopes that are important to neutralization are properlypresented in the vaccine constructs. While this later point might seemtrivial, there has heretofore been no simple way to learn whethercritical epitopes are destroyed during construction of subunit vaccinesor even during formulation of intact virus preparations.

Finally, we come to the often asked interesting question of whether itmatters that the libraries were prepared from patients who successfullycombated an infection as opposed to animals or people that simply havebeen immunized with viral antigens. Because a substantial fraction ofpatients in our cohort died, it is tempting to speculate that thesurvivors made antibodies that were related to their favorable clinicaloutcome. This is a difficult argument because so many factors contributeto patient survival, several of which have little to do with therobustness of the immune response. It simply should be said that naturalantibodies obtained from survivors can reasonably be expected to be atleast as good as, and perhaps better, than those obtained after simpleimmunization with inert antigens. At the very least, one can be certainthat the virus has been presented in a manner that allowed an immuneresponse appropriate to survival of the individual. Thus, we gainedinsight from this analysis about how the immunological repertoiresearches sequence space when, because of the virulence of the infectiousagent, time is short.

Materials and Methods

H5N1 Turkish cases—bone marrow recovery. Six H5N1 survivors providedbone marrow and serum for this study. All were diagnosed betweenDecember 2005 and January 2006. Descriptions of their diagnoses havebeen previously reported (Oner et al., (2006) N. Engl. J. Med.355:2179-2185). Briefly, most samples were nasopharyngeal swabs testedby ELISA, rapid influenza test, and/or real-time polymerase chainreaction in Turkey. Additionally, four of the six survivors were furtherverified by WHO laboratory testing in London. Following four and fivemonths post recovery bone marrow aspirates and serum from the sixsurvivors were collected, minimally processed in RNALater (Ambion) topreserve RNA integrity, and shipped frozen on dry ice to ourlaboratories. This study was reviewed and approved by both the TurkishMinistry of Health and the Yuzuncu Yil University, Van, Turkey. Writtenguardian consent was provided for all donors.

Antibodies Proteins and viruses. Hemagglutinin proteins were eitherpurchased from Protein Sciences (H5 Protein A/Vietnam/1203/2004, H1Protein A/New Calcdonia/20/99, H3 Protein A/Wisconsin/67/05) orgenerated by de novo synthesis (H1 Protein A/South Carolina/1/18) aseukaryotic codon optimized soluble secreted HA genes (DNA 2.0) and thensubcloned into pCI (Promega) for mammalian protein expression, sequenceverified, and then transfected into 293 Freestyle cells (Invitrogen)according to manufacturers guidelines. Briefly 20 μg of light chain and10 μg heavy chain encoding plasmid were combined with 1.0 m1293 fectinand incubated for 60 minutes. Following this preincubation the DNAmixture was combined with 3×10⁷ cells in 30 ml media for then and theresulting cell suspension was grown according to manufacturerssuggestion for 7 days. After seven days the secreted immunoglobulinswere purified from the culture supernatants using protein Achromatography (Calbiochem). The resulting purified antibodies werebuffer exchanged into sterile PBS using centrifugal size filtration(Centricon Plus-20) and their protein concentrations determined bycalorimetric BCA assay (Pierce).

Recombinant viruses were genetically engineered and produced asdescribed elsewhere (Fodor et al., (1999) J. Virol. 73:9679-9682).Additionally, Indonesia, Turkey, and Egypt were similarly made excepttheir HA genes were synthetically assembled using eukaryotic codonoptimized sequences (DNA 2.0). Inactivated viruses were made asdescribed elsewhere (Fodor et al., supra).

Serology: Hemagglutinin and viral ELISA. Recombinant HA proteins: H5Protein A/Vietnam/1203/2004 (Protein Sciences)—10 ng/well; RecombinantHA H1 Protein A/New Calcdonia/20/99 (Protein Sciences)—10 ng/well;Recombinant HA H3 Protein A/Wisconsin/67/05 (Protein Sciences)—10ng/well; H1N1 Virus A/New Calcdonia (BioSource)—70 ng/well; H3N2 VirusA/Panama/2007/99 (BioSource)—10 ng/well; FDA Influenza Virus Vaccine forH5N1 rgA/Vietnam/1203/2004 (CBER)— 10 ng/well.

ELISA plates were coated as indicated with either recombinanthemagglutinin protein or inactivated virus overnight incubation at roomtemperature. The next day plates were appropriately blocked (1% bovineserum albumin in PBS/0.05% Tween-20) and then 0.1 ml serum samples,diluted in blocking buffer, were incubated, washed, and detected using aperoxidase conjugated anti-human Fe antibody (Jackson Immuno) and TMBdetection (BioFX). Absorbance at 450 nm was read, data recorded, andreported herein.

Donor specific repertoire recovery. Between 2-2.5 ml of donor bonemarrow previously stored in 25 ml RNAlater (Ambion) was processed withTRI-BD (Sigma) according to manufacturers directions and then furtherprocessed to extract purified total RNA as described elsewhere (Barbaset al., (2001) Phage Display: A Laboratory Manual (Cold Spring HarborLab Press, Cold Spring Harbor, N.Y.)). Next mRNA was purified byOligotex spin column purification (Qiagen). Next random nonamer primedreactions and oligo dT reverse transcription reactions performed usingAccuscript (Stratagene) according to manufacturers directions.

For each donor the following was performed. For each of the 11 lambdalight chain families a single PCR amplification was performed withfamily specific V_(L) primers, a mixture of J_(L) primers, and primedwith 75 ng Oligo dT cDNA. Kappa recovery was similarly performed foreach of the six kappa light families, except that 75 ng of randomnonamer primed cDNA was used. For heavy chain recovery VH1/7, VH3, andVH4 were individually recovered, and VH 2,5, and 6 were pool amplifiedwith gene specific primers, a mixtures of J_(H) primers, and each wasprimed with 100 ng of random nonamer primed cDNA. Primers andamplification conditions were essential as described elsewhere usingPlatinum Pfx polymerase (Invitrogen) (ref). PCR products were minimallyprocessed by PCR Cleanup (Qiagen) quantitated by A₂₆₀. Heavy chainreactions were gel purified and then, if necessary, amplified again toproduce quantities sufficient for cloning.

Phage Library Construction.

Light chain cloning. Donor specific barcoded vectors and equimolar poolsof Kappa and lambda light chains were separately digested with NotI andBamHI and gel purified using (Qiagen). Library ligations were performedwith 200 ng of gel purified Kappa or Lambda inserts and 1 μg of gelpurified vector. Incubation is for at least one hour at RT or overnightat 14° C. Ligations were desalted using Edge BioSystem Perfroma spincolumns. 5 electroporations per library were done in 80 μl TG-1aliquots, each recovered in 1 ml SOC, pooled and outgrown for one hourat 37° C. A sample of each was taken for plating and used to determinethe total number of transformants. The remainder was transferred to 200ml 2YT+100 μg/ml Ampicillin+2% glucose and grown overnight at 37° C.Target number of transformants/library was at least 1×10⁶/μg vector DNA.Light chain library plasmids were then pelleted and the plasmidspurified using a Qiagen High Speed Maxiprep Kit.

Heavy chain cloning and phage production. Donor specific heavy chains(V_(H)1, V_(H) 3, V_(H) 4, and V_(H) 2, 5, 6 pool) and light chainlibrary collections were separately digested with a 40 Unit/μg DNA withSfiI and XhoI and gel purified (Qiagen). 5 μg of kappa and lambda lightchain libraries were separately ligated, overnight, with 1.2 μg of anequimolar mix of the four donor specific heavy chain preparations. Thelibrary ligations were spin column desalted (Edge BioSystem) and thentransformed in 16-20 electroporations per library. Processing todetermine the number of transformants is as described above. Phageproduction proceeded as described elsewhere. Following phage productionthe phage was harvested by PEG/NaCl precipitation and resuspended andstored in PBS containing 50% glycerol.

Panning and Clonal ELISAs. Panning and clonal ELISAs were performed asdescribed previously (Fodor, E. et al. J Virol. 1999; 73(11): 9679-82).

Microneutralization. Cross sub-type neutralization by antibodiesrecovered from survivors of avian influenza. Indonesia and Turkeyhemagglutinin genes were synthetically assembled using human codonoptimized sequences (DNA 2.0) and then used to generate recombinantengineered viruses. Recombinant influenza viruses were generated usingreverse genetics as previously described (Fodor, E. et al. J Virol.1999; 73(11): 9679-82). Briefly, 1 ug each of 10 plasmids wastransfected into 293 T cells in monolayer. Each transfection containedambisense plasmids (for the expression of both vRNAs and mRNAs) for theA/Puerto Rico/8/34/PA, PB1, PB2, NP, M, and NS segments, in addition tovRNA (pPOL1 type) and protein expression plasmids (pCAGGS type) forA/Vietnam/1203/04 HA and NA (pCAGGS expression plasmid was kindlyprovided by J. Miyazaki, Osaka University, Osaka, Japan) (Miyazaki, J.et al. Gene 1989; 79(2):269-77). Twenty hours following transfection,293T cells were resuspended in cell culture supernatant, and used toinoculate 10-day-old embryonated eggs.

Antibodies were screened for neutralizing activity against viruses asfollows. Two fold serial dilutions of each Mab were incubated with 100TCID₅₀ of virus in PBS at 370 for 1 h. Madin-Darby Canine Kidney cellmonolayers in 24 well plates were washed once with PBS and inoculatedwith virus-antibody mixtures. Following incubation for 1 h at 37° C. in5% CO₂, the inoculum was removed and monolayers were again washed oncewith PBS. Opti-MEM supplemented with 0.3% BSA, 0.01% FBS and 1 ug/mlTPCK-treated trypsin was added and cells were incubated for 72 h at 37°C. The presence of virus in cell culture supernatants was assessed by HAassays using 0.5% chicken red blood cells.

Cross-reaction IgG ELISA. Microtiter plates were coated with 0.1 ml ofthe following antigens diluted in coating buffer and incubated overnightat room temperature: 100 ng/ml H5N1 Vietnam 1203/04, 250 ng/ml H5N1Turkey/65596/06, 1 μg/ml H5N1

Indonesia/5/05, 700 ng/ml H1N1 New Calcdonia/20/99, 1 μg/ml H1N1 NorthCarolina/1/18, 100 ng/ml and H3N2 Wisconsin/67/05. Blocking was donewith 0.3 ml of blocking buffer (4% Non-fat dry milk in PBS/0.05%Tween-20). Following blocking antibodies diluted to 0.5 μg/ml in 2%non-fat dry milk blocking buffer were incubated for two hours at 4 C,washed, and later detected using a 1:3000 dilution of peroxidaseconjugated anti-human F_(c) antibody (Jackson ImmunoResearch) in 2%non-fat dry milk blocking buffer and standard TMB substrate detection(BioFX). Absorbance at 450 nm was read, data recorded, and reportedherein. (B) Relative ranking of antibodies by their ELISA signal tonoise ratios (−<2, +=2−<9, ++=9−<15, +++=≧15), on various proteins andminimal inhibitory concentration (MIC) in microneutralization assay.Suitable protocols can be found in Barbas C. et al. (2001) PhageDisplay, A Laboratory Manual (Cold Spring Harbor Laboratory Press).

Epitope Analysis of Hemagglutinin-Binding Fabs.

Biotinylation of HA Proteins: 100 ug of purified Hemagglutinin proteinis biotinylated at a 20:1 molar excess using Pierce No-Weigh PEO4 biotin(cat #21329) according to manufacturers instructions, incubated at roomtemperature for 1-3 hours with intermittent mixing and then incubatedovernight at 4 C. The excess biotin is removed by size exclusion spincolumn and exchanged into PBS.

Quantitation of Fabs: HA binding Fabs are purified by FPLC using Ni²⁺affinity chromatography, desalted to remove excess imidazole,concentrated, and quantitated by quantitative light chain ELISAs (BethelLabs, cat # E80-115-κ, and E80-116-λ) are performed according to themanufacturers instructions.

Sample set up: HA protein is bound to sensors and allowed to reach newbaseline. Next, sample and epitope binding standards are tested for HAsaturation using the conditions determined from kinetic analysis.Desalted, concentrated Fabs were evaluated for HA binding in a typicalrange of 0.5-16 ul in 200 ul sample diluent. Using the conditionsidentified in saturation testing, standard epitope binding antibodiesare first loaded on to HA coated biosensors. A new baseline isestablished and then the test samples at half saturation concentrationsare loaded on to the epitope saturated sensors. Antibodies are testedagainst all possible epitope recognition standards in this way. Thefollowing is a summary of the sample type and time the sensors are heldin each column of solution:

Column 1 Baseline Sample Diluent 1-2 minutes Column 2 HA BindingBiotinylated HA 5-15 minutes Column 3 Baseline Sample Diluent 1-2minutes Column 4 Saturation Diluted antibodies 5-15 minutes Column 5Baseline Sample Diluent 1-2 minutes Column 6 Sample binding Diluted testantibody 5-15 minutes Column 7 Baseline Sample Diluent 1-2 minutes

Increased interference shift above saturation levels indicates novelepitope recognition. Three possible results from this type of analysisare:

1) Complete blocking—No interference shift

2) Restoration of saturation—If dissociation of the standard occursduring the baseline after binding, sample binding that restores thesignal to saturation levels indicates binding to the same epitope

3) New epitope binding—Increased interference shift above saturationlevels

Kinetic Analysis of Hemagglutinin-Binding Fabs.

Biotinylation of HA Proteins: 100 ug of purified Hemagglutinin proteinis biotinylated at a 20:1 molar excess using Pierce No-Weigh PEO4 biotin(cat #21329) according to manufacturers instructions, incubated at roomtemperature for 1-3 hours with intermittent mixing and then incubatedovernight at 4 C. The excess biotin is removed by size exclusion spincolumn and exchanged into PBS.

Quantitation of Fabs: HA binding Fabs are purified by FPLC using Ni²⁺affinity chromatography, desalted to remove excess imidazole,concentrated, and quantitated by quantitative light chain ELISAs (BethelLabs, cat # E80-115-κ, and E80-116-λ) are performed according to themanufacturers instructions.

Kinetic Analysis: Kinetic analysis is performed on a range of sampleconcentrations that are empirically determined. The first range istypically 15 nM-500 nM in serial 2 fold dilutions and the samples areincubated with biosensors coated with HA protein for up to 15 minutes,then incubated in sample diluent for up to 1 hour. All of these stepsare done with sample plate rotation at 1500 RPM. Association is measuredduring the Fab incubation with the HA-coated biosensors and dissociationis measured in the sample diluent incubation following binding. Thefollowing is a summary of the sample type and time the sensors are heldin each column of solution:

Column 1 Baseline Sample Diluent 1-2 minutes Column 2 HA BindingBiotinylated HA 5-15 minutes Column 3 Baseline Sample Diluent 1-2minutes Column 4 Association Diluted antibodies 5-15 minutes Column 5Dissociation Sample Diluent 15-180 minutes

Data analysis using the Forte Bio Kinetic Analysis software providesestimates of on and off rates with r² values. A value is deemed to bereportable if of high confidence with r² values >0.95. The k_(D) is thenaccepted as the affinity of the molecule.

Viral microneutralization. The VN activity of MAbs was measured asfollows. MAb dilutions (50 ml) in ISC-CM—0.1% BSA, eight replicates perdilution, were dispensed into 96-well flat-bottom tissue culture plates.PR8 (50 ml) in ISC-CM—0.1% BSA; (100 TCID50) were added to each well,and the plates were incubated for 1 h at 37° C. MDCK cells were thenadded to each well (25 ml ISC-CM—0.1% BSA containing 2 3 106 cells/ml),and the plates were incubated for 8 to 14 h to permit MDCK cells toadhere. The medium was then flicked out and replaced with 200 ml ofantibody-free ISC-CM—0.1% BSA supplemented with trypsin (2.5% trypsin[Whittaker Bioproducts Inc.]) at a final dilution of 1/3,000; (8 mg/ml).After another 2.5 days of incubation, culture supernatants were testedfor the presence of virus by HA titer determination. The MAbconcentration at which 50% of the cultures were protected from infectionwas computed by interpolation and taken as the MAb VN activity. Notethat low concentrations indicate high VN activity (Mozdzanowska, K. etal. (1997) J. Virol. 71, 4347-4355).

Example 2 Generating Universal Influenza Vaccines

The goal of vaccine design against heterogeneous pathogens is toidentify and design effective and broadly protective antigens. In thecase of influenza, considerable historical efforts have gone into theempirical testing of conserved linear sequences and regions with littlesuccess. A plausible reason for these failures is a lack of knowledgethat focused responses against antigenic test articles are actual bonafide productive sites for neutralization of an antigen on the pathogenin the setting of an actual infection. For influenza one would be expectto find these bona fide solutions within the repertoires of survivors ofan influenza infection. In our case we have demonstrated that severalrelated antibodies amongst a large collection of antibodies, derivedfrom an H5N1 influenza survivor, (see Table 4 above) are capable ofbroadly neutralizing several subtypes of Influenza. These antibodiesneutralize influenza through a novel mechanism that does not involveclassical inhibition of hemagglutination, which has now been confirmedand delineated at a structural level by two additional and independentgroups. Collectively, we expect that the design and assessment ofvaccines utilizing such cross neutralizing antibodies derived from bonafide survivors would aid in the design and validity of cross reactive or“universal” influenza vaccines.

Specifically cross neutralizing monoclonal antibodies can be used in thedesign and validation of vaccine production processes that maintain orenhance the quality and antigenicity of cross neutralizing epitopes incurrent and future manufactured vaccines. Assuming that antibody bindingto vaccine is reflective of structural integrity and antigenicpotential, one would assess binding of cross neutralizing antibodies,such as Ab-1 (see Table 4 above) to such vaccine process derivatives toquantitatively assess their cross neutralizing potential.

To maximize the responses toward these universal epitopes one wouldcreate derivatives to increase immunogenicity towards these universalepitopes. In this case the resulting antigen would again be tested toinsure that not only the efficiency of binding to target was maintained,but that a directed immunogenicity was accomplished. This would eitherinvolve determining the specific universal neutralizing titers containedin the serum from immunized individuals or test animals, likely bycompetitive ELISA against Ab-1 (or related antibody) from Table 4. As anin vitro surrogate, one would combine the antigen-antibody binding datawith that of an in vitro or in silico predictive model forimmunogenicity. To further direct responses to the universal epitope onemay deimmunize known non-neutralizing hemagglutinin epitopes

It reasonable to extend this antibody for the design and validation ofengineered recombinant hemagglutinin chimeras, fragments, andconformational mimics. For instance, it is well established thatinfluenza contains many immunodominant epitopes that give rise tonon-neutralizing responses. Utilizing the cross protective antibodies itis possible to assess whether antigen variants of vaccines that havebeen partially or fully deimmunized for these immunodominantnon-neutralizing epitopes have maintained or created enhancedrecognition of the universally protective epitopes.

Additional ways to guide a specific response to a distinct epitope is tosimply remove non-neutralizing and non-conserved regions from therecombinant vaccine design. As an example we would remove the HA1, orHA0 globular sialic binding domain of hemagglutinin to leave the moreconserved stem region of hemagglutinin as the principal target for animmune response. As sequence space does not strictly correlate tophysical space, this will require the removal of middle coding regionsfor proteins to create such aglobular constructs. Further as more of theglobular domain is removed this will cause residues that are normallyembedded within the protein structure to be exposed. These residues thatare not normally solvent exposed may need to be mutagenized anddeimmunized to residues that are better suited structurally or morecompatible to solvent exposure. Similar to efforts described above, wewould use the antibodies identified previously to insure the integrityof these cross protective epitopes.

From these aglobular vaccine designs, one could minimize the antigenepitopes and even remove them from the context of hemagglutinin tocreate a conformational cross specific antigen.

The strategies outlined above detail methods to guide a response to aminimized neutralizing epitope or element. From the knowledge of suchminimized elements, which are likely be conformationally dependent andexist within discontinuous sequence space, it would be possible torecreate the conformational neutralizing epitope in a combinatorialfashion within a smaller polypeptide, as described previously (seeHorowitz et al., Combinatorial Libraries of Conformationally ConstrainedPolypeptide Sequences, PCT/US2008/050877) where the proximal placementof discontinuous epitopes alone, or in the context of designedstructural support, can regenerate the essential properties ofconformational epitopes.

In such a design we would take the conformation epitope and express themon hemagglutinin related and unrelated structural scaffolds, or even asa collection of conformational epitopes within a library that could beselected by conformationally dependent antibodies such as Ab-1.

The reduction of discontinuous epitopes to a conformational epitopewould result in an even smaller sized peptide immunogen than thatpossible with traditional protein engineering. Furthermore thesestructural epitopes may be further enhanced, reduced in size, orsubstituted through the use of nonpeptide mimetics. In any event, any ofthese conformational derivatives or mimics would be validated by theAb-1, Ab-1 related antibody, or corresponding antibody to the influenzavirus of choice.

Methods and materials. Influenza fusion epitope spore vaccine targets.

-   -   1. Mammalian expression of target as secreted protein or on        mammalian cell.        -   a. Express stem (HA2 only)        -   b. aglobular HA0        -   c. aglobular HA1/HA2        -   d. aglobular HA1/topless HA2    -   2. Detect conformational epitope with A6-related antibody of        secreted protein or on mammalian cell    -   3. Transfer successful stem or aglobular antigen to spore        expression    -   4. Test for spore binding with A6-related antibody    -   5. Immunize mice

Example 3 Increasing the Potency and Spectrum of Cross SubtypeNeutralizing Antibodies

As mentioned previously, the group of cross subtype neutralizingantibodies that are partially represented by Ab-1, 2, & 3 contain verydistinct and seemingly requisite heavy chain mutations within CDR2 andframework 3 (FR3), yet remarkably little to no diversity within CDR3.Considering the shear number of clones that were identified with thesehallmark sequences, all of which were restricted to a 1-e, or 1-e likeframeworks, leads one to suspect that this broad spectrum activity isprincipally driven by the this specific heavy chain framework and theCDR2 and Framework 3 (FR3) mutations. Recently, two groups haveconfirmed this at a structural level by analyzing co-crystals ofhemagglutinin and other broad spectrum antibodies that utilize the 1-elike, 1-69 germline framework (Kashyap et al. supra; Throsby et al.,PLoS ONE 3(12): e3942). In each instance the predominant binding wasdriven by CDR2 and FR3 sequence corresponding to the areas described byKashyap et al. (supra). To identify minimal binding elements for thisbroad specificity one would begin by serially reverting back each of theCDR2 and FR3 mutations to germline and assess broad subtype influenzabinding. In the case of CDR3, alanine scanning would be used to furtherdefine the crossreactive minimal essential elements.

Upon learning the range of sequence involved in broad specificitybinding we would use methods of mutagenesis to create improved mutantsfor testing either individually or amongst a collection in a library.Methods commonly used to introduce mutations could be saturationmutagenesis at sites responsible for binding or error-prone PCRmutagenesis throughout the regions known to be responsible for binding.Similarly, the previously mentioned mutagenesis methods could be appliedto other areas of the heavy chain that may influence recognition in amore global manner.

Once these 1-e or 1-e like optimized clones were identified we nextutilize recombinant methods to graft these defined minimal elements ontoother related and unrelated heavy chain frameworks. This gives us theability to explore additional optimized solutions under differentcontexts that may be superior to the original. As a next step theseminimal elements would be modeled and/or grafted onto other related andunrelated proteins. The success of these efforts could provide superiorpharmacological agents and even avenues leading to minimized orconstrained peptides that either present or mimic the crossreactivebinding motif mimetic. Success at this stage would then be extended intothe area of nonpeptide-mimetics.

Finally, searching the sequence databases for other related antibodiesrevealed numerous anti-infectious antibodies, suggesting the 1-e or 1-elike framework may function as a first line defense against infectiousorganisms and viruses. As such it is presumable that 1-e or 1-e likerepertoires would be ideal sources for de novo identification ofanti-infectious antibodies that could be developed similar to theoutlined steps for the Ab-1 and related antibodies for influenza.

Materials and methods. 1-69/1-e anti-idiotype antibodies and vaccines.

-   -   1. Pan minimized framework element antibody for specific        reagent.    -   2. Administer anti-idiotype antibody in presence or absence of B        cell stimulating agent to expand anti-influenza repertoire.    -   3. Measure anti-influenza titer (ex vivo from PBMC or bone        marrow).

Example 4 Inducing 1-e and 1-e-Like Anti-Influenza Antibodies

Inducing the proliferation of memory B cells causes the proliferationand secretion of the specific antibodies the stimulated B cells.Presumably upon learning the minimal binding elements required for crosssubtype hemagglutinin binding one could use this element as a selectiontool to identify anti-idiotype antibodies. The administration of such aframework and mutation specific anti-idiotype antibodies would result inthe expansion of these broad specific memory B cells and the serologicalincrease of these anti-influenza antibodies in the setting ofprophylaxis or treatment of disease.

Again, as searching the sequence databases for other related antibodieshas revealed numerous anti-infectious antibodies, suggesting the 1-e or1-e like framework may function as a first line defense againstinfectious organisms and viruses. It is presumable that expansion of 1-eor 1-e like anti-idiotype repertoires would be ideally suited forprotection or treatment of infectious disease.

Agents to induce or produce broadly specific antibodies (1-69/1-e andrelated frameworks). Agents include rearranged Vh for delivered as genetherapy (in vivo & ex vivo), engineered transcriptional activators of Vhspecific genes. Such agents would be useful for influenza (antiviral)treatment/prophylaxis; as an adjuvant for (antiviral) prophylaxis; exvivo selection (and possible expansion) of Vh specific B cells fortreatment and prophylaxis; influenza epitopes for Vh specificinduction/production; 1-69/1-e and related anti-idiotypeantibodies/surrobodies to expand Vh specific memory response (mayinclude costimulatory agent on a surrobody or separate administration);vaccines directed to 1-69/1-e frameworks to induce proliferation andproduction of 1-69/1-e and related antibodies; and any combination ofthe above.

Example 5 Co-Administration of Vaccine and Antibody to Increase Potencyand Spectrum of Protection

Complexes of antibody and antigen are known to potently induce responsesagainst numerous microbial proteins and other proteins in animals. Onepossible explanation is that a forced uptake of the vaccine antibodycomplex occurs by Fc receptors on antigen presenting cells. Complexes ofcross reactive antibodies, such as Ab-1 with seasonal vaccines wouldallow for increases in potency from year to year and because Ab-1 andthe related antibodies recognize numerous hemagglutinin antigens,obviates the need to recreate new antibodies when new viral isolates areselected for each seasons Influenza vaccine. Furthermore, as theseantibodies are directed to conserved neutralizing regions they mayactually direct a more effective protective response towards thesecritically conserved susceptible regions when complexed with antigen. Asdescribed previously, the vaccine may be a traditional live or killedvirus, recombinant protein or protein fragment, or even minimizedpeptide or non-peptidic conformationally epitope complexed with anantibody, antibody fragment or derivative, or surrobody.

Although in the foregoing description the invention is illustrated withreference to certain embodiments, it is not so limited. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

All references cited throughout the specification are hereby expresslyincorporated by reference in their entirety.

1. A molecule, which is an antibody or antibody-like molecule, whereinsaid molecule (i) neutralizes more than one subtype and/or more than oneisolate of an influenza A virus, (ii) binds to a hemagglutinin (HA)antigen of the virus, and (iii) does not inhibit hemagglutination. 2.The molecule of claim 1, which is a polypeptide comprising a VpreBsequence and/or a λ5 sequence.
 3. The molecule of claim 1, which is apolypeptide comprising a VpreB sequence fused to a λ5 sequence.
 4. Themolecule of claim 1, which is a κ-like surrogate light chain (SLC)construct comprising a Vκ-like and/or a JCκ sequence.
 5. The molecule ofclaim 1, which is an antibody.
 6. The molecule of claim 1 which iscross-reactive with at least two HA antigens selected from the groupconsisting of H1, H2, H3, H5, H6, H7, H8 and H9.
 7. The molecule ofclaim 1 which is cross-reactive with at least two HA antigens selectedfrom the group consisting of H1, H2, H3, H5, and H9.
 8. The molecule ofclaim 1 which binds to an epitope of an H1 subtype of the HA antigen. 9.The molecule of claim 1 which binds to an epitope of an H5 subtype ofthe HA antigen.
 10. The molecule of claim 1 which binds to an epitope ofan H3 subtype of the HA antigen.
 11. The molecule of any one of claims8, 9, or 10 wherein the epitope is displayed on the surface of aninfluenza A virus.
 12. The molecule of any one of claims 8, 9, or 10which neutralizes at least one of the H5, H3, and H1 influenza A virussubtypes.
 13. The molecule of any one of claims 8, 9, or 10 whichneutralizes more than one isolate of an H5 and/or H3 and/or H1 subtypeof an influenza A virus.
 14. The molecule of claim 1, which does notprevent the globular head region of the influenza A virus from bindingthe surface of a cell.
 15. The molecule of claim 1 wherein at least oneof said viruses has the ability to infect humans.
 16. The molecule ofclaim 1 wherein at least one of said isolates has been obtained from ahuman subject.
 17. The molecule of claim 1 wherein at least one of saidisolates has been obtained from a non-human animal.
 18. The molecule ofclaim 17 wherein said non-human animal is a bird.
 19. The molecule ofclaim 18 wherein said bird is a wild-fowl or a chicken.
 20. The moleculeof claim 1 which binds to an H1 HA antigen.
 21. The molecule of claim 20which binds to at least one additional HA antigen.
 22. The molecule ofclaim 21 wherein said additional HA antigen is selected from the groupconsisting of H2, H3, H5, H6, H7, H8 and H9.
 23. The molecule of claim21 which additionally binds HA antigen H5.
 24. The molecule of claim 21which additionally binds HA antigens H3 and H9.
 25. The molecule ofclaim 21 which additionally binds HA antigens H3, H5, and H9.
 26. Anantibody or antibody-like molecule which binds essentially the sameepitope as the epitope for an antibody or antibody-like moleculecomprising a heavy chain polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:4, SEQ ID NO:45, SEQ IDNO:9, and SEQ ID NO:61; or a consensus or variant sequence based uponsaid amino acid sequences.
 27. The antibody or antibody-like molecule ofclaim 26 which binds essentially the same epitope as the epitope for anantibody or antibody-like molecule comprising a light chain polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158, SEQ ID NO:159,and SEQ ID NO:160; or a consensus or variant sequence based upon saidamino acid sequences.
 28. An antibody or antibody-like molecule whichbinds essentially the same epitope as the epitope for an antibody orantibody-like molecule comprising a heavy chain polypeptide comprisingan amino acid sequence having the formula:X₁-X₂-Q-L-V-Q-S-G-X₃-E-V-X₄-K-P-G-X₅-S-V-X₆-X₇-S-C-K-X₈-S-G-G-X₉-F-S-S-Y-A-X₁₀-X₁₁-W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X₁₂-G-I-I-X₁₃-X₁₄-F-G-T-T-X₁₅-N-Y-A-Q-K-F-Q-G-R-X₁₆-T-X₁₇-T-A-D-X₁₈-X₁₉-T-S-T-A-Y-M-E-L-S-S-L-R-S-X₂₀-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X₂₁-X₂₂-L-D-Y-W-G-X₂₃-G-T-X₂₄;or a consensus or variant sequence based upon said amino acid sequences,or a fragment thereof; wherein X₁ is Q or E; X₂ is V or M; X₃ is A or T;X₄ is K or Q; X₅ is S or A; X₆ is K or R; X₇ is V or L; X₈ is A, T or V;X₉ is T, S or A; X₁₀ is I or V; X₁₁ is S or T; X₁₂ is G or A; X₁₃ is Por G; X₁₄ is I or M; X₁₅ is A or T; X₁₆ is V or L; X₁₇ is I, L, or M;X₁₈ is K or E; X₁₉ is S, L or M; X₂₀ is E or D; X₂ is S, T or N; X₂₂ isS or T; X₂₃ is Q, K, G or R; and X₂₄ is L, T or M.
 29. The antibody orantibody-like molecule of claim 28 which binds essentially the sameepitope as the epitope for an antibody or antibody-like moleculecomprising a light chain polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:71, SEQ ID NO:140, SEQID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160; or aconsensus or variant sequence based upon said amino acid sequences. 30.An antibody or antibody-like molecule comprising a heavy chainpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:4, SEQ ID NO:45, SEQ ID NO:9, and SEQ ID NO:61;or a consensus or variant sequence based upon said amino acid sequences.31. The antibody or antibody-like molecule of claim 30 furthercomprising a light chain polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:71, SEQ ID NO:140, SEQID NO:81, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160; or aconsensus or variant sequence based upon said amino acid sequences. 32.An antibody or antibody-like molecule comprising a heavy chainpolypeptide comprising an amino acid sequence having the formula:X₁-X₂-Q-L-V-Q-S-G-X₃-E-V-X₄-K-P-G-X₅-S-V-X₆-X₇-S-C-K-X₈-S-G-G-X₉-F-S-S-Y-A-X₁₀-X₁₁-W-V-R-Q-A-P-G-Q-G-L-E-W-M-G-X₁₂-G-I-I-X₁₃-X₁₄-F-G-T-T-X₁₅-N-Y-A-Q-K-F-Q-G-R-X₁₆-T-X₁₇-T-A-D-X₁₈-X₁₉-T-S-T-A-Y-M-E-L-S-S-L-R-S-X₂₀-D-T-A-V-Y-Y-C-A-R-G-S-Y-Y-Y-E-X₂₁-X₂₂-L-D-Y-W-G-X₂₃-G-T-X₂₄;or a consensus or variant sequence based upon said amino acid sequences,or a fragment thereof; wherein X₁ is Q or E; X₂ is V or M; X₃ is A or T;X₄ is K or Q; X₅ is S or A; X₆ is K or R; X₇ is V or L; X₈ is A, T or V;X₉ is T, S or A; X₁₀ is I or V; X₁₁ is S or T; X₁₂ is G or A; X₁₃ is Por G; X₁₄ is I or M; X₁₅ is A or T; X₁₆ is V or L; X₁₇ is I, L, or M;X₁₈ is K or E; X₁₉ is S, L or M; X₂₀ is E or D; X₂₁ is S, T or N; X₂₂ isS or T; X₂₃ is Q, K, G or R; and X₂₄ is L, T or M.
 33. The antibody orantibody-like molecule of claim 32 further comprising a light chainpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:71, SEQ ID NO:140, SEQ ID NO:81, SEQ ID NO:158,SEQ ID NO:159, and SEQ ID NO:160; or a consensus or variant sequencebased upon said amino acid sequences.
 34. The antibody or antibody-likemolecule of any one of claims 26 to 33 which (i) neutralizes more thanone subtype and/or more than one isolate of an influenza A virus, (ii)binds to a hemagglutinin (HA) antigen of the virus, and (iii) does notinhibit hem agglutination.
 35. A composition comprising a moleculeaccording to any one of claims 1 to
 25. 36. A composition comprising anantibody or antibody-like molecule according to any one of claims 26 to34.
 37. A molecule comprising an antibody heavy chain variable domaincomprising at least one substitution in the surface exposed clusterdetermined by amino acid positions 52A, 53, 73, and 74, following Kabatamino acid numbering, wherein said molecule is capable of binding to andneutralizing a viral antigen.
 38. The molecule of claim 37, comprising asubstitution at least one of amino acid positions 52A, 53, 73, and 74.39. The molecule of claim 37, comprising a substitution at all of aminoacid positions 52A, 53, 73, and
 74. 40. The molecule of claim 39,further comprising a substitution at amino acid position
 57. 41. Themolecule of claim 39 comprising P52G, 153M, L73E, and S74L/Msubstitutions.
 42. The molecule of claim 41 additionally comprising anA57T substitution.
 43. The molecule of claim 42 additionally comprisinga substitution at least one of amino acid positions 24, 34, 35 and 50.44. The molecule of claim 43 comprising substitutions at all of aminoacid positions 24, 34, 35 and
 50. 45. The molecule of claim 44comprising V24T, W34V, G35T and S50A substitutions.
 46. The molecule ofany one of claims 37 to 45 wherein the heavy chain variable domainsequence is from the V_(H) 1e germ-line heavy chain.
 47. The molecule ofclaim 46 wherein the rest of the heavy chain variable domain sequenceretains the sequence of the V_(H) 1e germ-line heavy chain.
 48. Themolecule of claim 46 wherein the V_(H) 1e germ-line heavy chain variabledomain comprises at least one additional conservative substitution. 49.The molecule of any one of claims 38 to 48, further comprising a lightchain sequence.
 50. The molecule of claim 49 wherein the light chainsequence is an antibody λ or κ light chain sequence.
 51. The molecule ofclaim 49 wherein the light chain sequence is a surrogate light chainsequence.
 52. The molecule of claim 51 wherein the surrogate light chainsequence comprises a VpreB sequence and/or a λ5 sequence.
 53. Themolecule of claim 52, wherein the surrogate light chain sequencecomprises a VpreB sequence fused to a λ5 sequence.
 54. The molecule ofclaim 51, wherein the surrogate light chain sequence is a κ-likesurrogate light chain (SLC) construct comprising a Vκ-like and/or a JCκsequence.
 55. The molecule of any one of claims 37 to 54, wherein theviral antigen is selected from the group consisting of viral antigensfrom influenza viruses, HIV-1, HIV-2, HTLV-I and -II viruses, SARScoronavirus, herpes simplex virus, Epstein Barr virus, cytomegalovirus,HCV, HAV, HBV, HDV, HEV, toxoplasma gondii virus, treponema pallidiumvirus, human T-lymphotrophic virus, encephalitis virus, West Nile virus,Dengue virus, Varicella Zoster Virus, rubeola, mumps, and rubella. 56.The molecule of claim 55 wherein the viral antigen is from an influenzavirus or an HIV-1 or HIV-2 virus.
 57. A vaccine effective against aninfluenza A virus, comprising a peptide or polypeptide functionallymimicking a neutralization epitope of a molecule according to any one ofclaims 1 to
 36. 58. A vaccine effective against a viral antigen,comprising a peptide or polypeptide functionally mimicking aneutralization epitope of a molecule according to any one of claims 37to
 57. 59. A method for identifying an antibody capable of neutralizingan isolate of an H5 influenza A virus and/or an isolate of an H1influenza A virus; or a subtype of an H5 influenza A virus and/or asubtype of an H1 influenza A virus, comprising identifying, in anantibody library, antibodies that react with both an H5 isolate and/oran H1 isolate; or an H5 subtype and/or an H1 subtype, and subjecting theantibodies identified to successive alternating rounds of selection,based on their ability to bind said H5 and/or H1 isolates or HAproteins; or said H5 and/or H1 subtypes or HA proteins, respectively.60. A collection of sequences shared by the neutralizing antibodiesidentified by the method of claim
 59. 61. A collection of sequencescomprising one or more of the unique heavy and/or light chain sequencesshown in Table 2 or a consensus or variant sequence based on saidsequences.
 62. A neutralizing antibody identifiable by the method ofclaim 59, or a fragment thereof.